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Arch Microbiol (2010) 192:409425 DOI 10.

1007/s00203-010-0563-y

MINI-REVIEW

Advances in molecular detection of Aspergillus: an update


M. Z. Abdin Malik M. Ahmad Saleem Javed

Received: 20 February 2009 / Revised: 1 November 2009 / Accepted: 10 March 2010 / Published online: 1 April 2010 Springer-Verlag 2010

Abstract Filamentous cosmopolitan fungi of the genus Aspergillus can be harmful in two ways, directly they can be opportunistic pathogens causing aspergillosis and indirectly due to aatoxin production on food products which can lead to aatoxicosis. Therefore, a number of methods have been proposed so far for detection of the fungi with lowest possible concentration at the earliest. Molecular methods such as PCR and/or in combination with certain techniques have been found to be useful for Aspergillus detection. We discuss here various technologies that have emerged in recent years and can possibly be used for the molecular detection of Aspergillus in an efcient way. These methods like RSIC, C-probe, and inversion probe with pyrosequencing or direct ss/dsDNA detection have been used for the identication of fungal or bacterial pathogens and thus formulate a gold standard for Aspergillus detection. Keywords Aspergillus Molecular detection PCR Polymerase chain reaction-enzyme immunoassay DNA ngerprinting Microarray technique Retrotransposon insertion-site context typing STRAf ELISA

Introduction Contamination with mycotoxins is a major problem of food and feeds storage. Mycotoxins are secondary metabolites produced by lamentous fungi. These toxic extrolites are not crucial for sustaining the life; instead, it is believed that they provide a competitive edge over other non-toxigenic molds and bacteria. Contamination of food and animal feeds with mycotoxin leads to adversely effect on human health also and leads to economic losses. Mycotoxins contaminate approximately 2550% of the total crops harvested, and since molds best thrive in tropical regions, they damage about 80% of the crops. There are about 300 different mycotoxins, but only 20 of them are known to have toxic effect on human when ingested along with contaminated food (Konietzny and Greiner 2003; Table 1). Mycotoxins like Aatoxins (AF), Ochratoxins (OT), Zeralenone (ZEN), Trichothecenes, and Fumonisins (F) are the major mycotoxins inuencing the public health and agriculture. World Health Organization-International Agency for Research on Cancer has categorized the mycotoxins according to its carcinogenic nature on humans. Aatoxins have been classied as the most carcinogenic mycotoxin and placed under Group I type while OT and F have been placed under Group 2B and trichothecenes have been kept under non-carcinogenic category of Group 3 (Hussein and Brasel 2001; WHO IARC 1993a, b). Aatoxins are produced by a large number of Aspergillus species, basically by three phylogenetically distinct sections. The main producers are A. avus, A. parasiticus, A. nomius, A. pseudotamarii, A. parvisclerotigenus, and A. bombycis of section Flavi. A. ochraceoroseus and A. rambellii from section Ochraceorosei and Emericella astellata, E. venezuelensis from section Nidulatans also

Communicated by Axel Brakhage. M. Z. Abdin M. M. Ahmad Department of Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi 110062, India S. Javed (&) Department of Biochemistry, Faculty of Science, Jamia Hamdard, New Delhi 110062, India e-mail: saleemjaved70@yahoo.co.in

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410 Table 1 List of 20 mycotoxins responsible for food spoilage and health problems
Mycotoxin Aatoxin Type B (B1, B2) Producer fungal species A. avus, A. parasiticus, A. tamarii, A. pseudotamarii, A. bombycis, A. parvisclerotigenus, A. nomius, A. minisclerotigenes, A. oryzae, A. toxicarius, A. versicolor, A. rambellii, A. arachidicola, A. ochraceoroseus, Emericella astellata, E. venezuelensis A. parasiticus, A. nomius, A. bombycis, A. pseudotamarii, A. terreus, A. versicolor, A. arachidicola, A. toxicarius, A. minisclerotigenes A. avus, A. versicolor, A. ochraceoroseus, A. rambellii, A. chevalieri, A. ruber, A. amstelodami, E. nidulans, E. rugulosa, E. venezuelensis A (OTA) Affected commodities

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References

Cotton seed, peanuts, peanut butter, Pildain et al. (2008), Foongpea, sorghum, rice, pistachio, Cunningham et al. (2006), Murphy maize, oilseed rape, maize our, et al. (2006), Samson et al. (2006), Frisvad and Samson (2004a), sunower seed, gs, spices, meats, dairy products, fruit juices Frisvad et al. (2004a), Konietzny (apple, guava) and Greiner (2003), Atalla et al. (2003), Hussein and Brasel (2001), Ito et al. (1995) Peanuts, cotton seed, sunower seed, tree nuts, pistachio, peanut butter, maize our, pea, cereals, corn, gs, meats, spices, dairy products, fruit juices (apple, guava) Peanuts, maize, peanut butter, pistachio, cotton seed, oilseed rape, tree nuts, sunower seed, soybeans, pea, cereals, gs Pildain et al. (2008), FoongCunningham et al. (2006), Murphy et al. (2006), Samson et al. (2006), Frisvad et al. (2005b), Somashekar et al. (2004), Atalla et al. (2003), Konietzny and Greiner (2003) Foong-Cunningham et al. (2006), Murphy et al. (2006), Frisvad and Samson (2004a), Frisvad et al. (2004a), Konietzny and Greiner (2003)

G (G1, G2)

Sterigmatocystin

Ochratoxin

Cereals (wheat, oats, barley), beans, Batista et al. (2009), FoongA. westerdijkiae, A. niger, soybeans, pea, nuts, coffee, cocoa Cunningham et al. (2006), Murphy A. ostianus, A. sclerotiorum, A. carbonarius, A. steynii, beans, grapes, g, sunower seed, et al. (2006), Samson et al. (2006), Frisvad et al. (2005b), Frisvad et al. A. sulphureus, A. sclerotionum, sugarcane, meats, dried fruits, (2004b) A. lacticoffeatus, A. sclerotioniger, spices, vinegar, wine P. verrucosum A. westerdijkiae, A. alliaceus, Cereals, beans, nuts, cocoa beans, A. sclerotiorum, A. sulphureus, dried fruits, spices, wine A. petrakii, Penicillium virdicatum, P. cyclopium, A. albertensis, A. auricomus, A. wentii A. ochraceus Wheat, oats, barley, maize, nuts, coffee, meats, dried fruits Batista et al. (2009), Murphy et al. (2006), Frisvad et al. (2004b), Bennett and Klich (2003), Hussein and Brasel (2001) Murphy et al. (2006), Frisvad and Samson (2004b), Bennett and Klich (2003) Murphy et al. (2006), Lillard-Roberts (2004), Atalla et al. (2003), Bennett and Klich (2003), Logrieco et al. (2003), Konietzny and Greiner (2003), Tomczak et al. (2002), El-Banna et al. (1984) Murphy et al. (2006), Lillard-Roberts (2004), Bennett and Klich (2003), Atalla et al. (2003), Logrieco et al. (2003), Hussein and Brasel (2001)

B (OTB)

C (OTC)

Trichothecenes

Deoxynivalenol (DON)

Fusarium graminearum, Barley, potatoes, wheat, silage, F. sambucinum, F. culmorum, maize, soybean, rye, sunower, F. sporotrichiodies, F. solani, porcessed grains, cookies, cornmeal Aspergillus parasiticus, A. terreus, A. oryzae, A. versicolor, Microdochium nivale F. solani, F. sporotrichiodes, F. acuminatum, F. lateritium, F. chlamydosporum, F. rigidiusculum, F. poae, F. sambucinum, A. oryzae, A. parasiticus F. solani, F. chlamydosporum, F. poae, F. sporotrichiodes, F. acuminatum, F. sambucinum Cereal crops (wheat, barley, oats, and rye), g, potato, processed grains (malt, beer, bread), silage

T2 toxin

HT2 toxin

Murphy et al. (2006), Lillard-Roberts Potato, maize, wheat, barley, oat, rye, processed grains (malt, beer), (2004), Bennett and Klich (2003), g, silage Logrieco et al. (2003), Konietzny and Greiner (2003), El-Banna et al. (1984) Potato, maize, wheat, sunower, corn, oat, g, processed grains, silage Murphy et al. (2006), Lillard-Roberts (2004), Bennett and Klich (2003), Logrieco et al. (2003), Hussein and Brasel (2001)

Diacetoxyscirpenol Gibberella intricans, F. equiseti, (DAS) F. solani, F. chlamydosporum, F. lateritium, F. sporotrichiodes, F. poae, F. sambucinum, F. acuminatum Nivalenol (NIV) F. tricinctum, F. poae, F. crookwellense, F. equiseti, F. graminearum, F. culmorum, F. sambucinum, A. parasiticus, A. versicolor

Wheat, cereals, maize, barley, corn, Murphy et al. (2006), Lillard-Roberts oat, rye, processed grains (malt, (2004), Bennett and Klich (2003), beer) Atalla et al. (2003), Logrieco et al. (2003), Konietzny and Greiner (2003), Tomczak et al. (2002) El-Banna et al. (1984)

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Mycotoxin Fumonisin Type B1 Producer fungal species F. nygamai, F. verticillioides, F. culmorum, F. avenaceum, F. roseum, F. proliferatum, F. acutatum, F. phyllophilum F. acutatum, F. pseudocircinatum, F. proliferatum, F. verticillioides, F. acutatum, A. niger Affected commodities References

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Maize, corn and products, asparagus spears, Murphy et al. (2006), Bennett and Klich garlic bulbs, wheat, rice (2003), Konietzny and Greiner (2003), Fotso et al. (2002), Hussein and Brasel (2001) Wheat, rice, maize, corn, sorghum, asparagus crown, onion, garlic, coffee beans Frisvad et al. (2007), Murphy et al. (2006), Lillard-Roberts (2004), Bennett and Klich (2003), Fotso et al. (2002), Hussein and Brasel (2001) Srensen et al. (2007), Murphy et al. (2006), Lillard-Roberts (2004), Bennett and Klich (2003), Fosto et al. (2002), Tomczak et al. (2002)

B2

Moniliformin (MON)

F. chlamydosporum, F. proliferatum, Cereals, maize, barley, oats, rice, pepper, F. monoliforme, F. oxysporum, ax, soybeans, millet, sorghum, barley, F. equiseti, F. begoniae, corn, wheat F. phyllophilum, F. subglutinans, F. avenaceum, F. tricinctum, F. pseudocircinatum, Microdochium nivale F. graminearum, F. culmorum, F. oxysporum, F. sporotrichiodes, F. moniliforme, F. avenaceum, F. crookwellense, F. equiseti, F. heterosporum, A. oryzae Penicillium commune, P. cyclopium, P. griseovum, P. camembertii, P. dipodomyicola, A. avus, A. tamarii, A. fumigatus, A. phoenicis, A. parvisclerotigenus, A. oryzae, A. caelatus, A. toxicarius A. pseudotamarii, A. minisclerotigenes Aspergillus clavatus, A. terreus, A. giganteus, Penicillium patulum (urticae), P. expansum, P. claviforme, P. griseovum, Byssochlamys nivea, Paecilomyces variotii

Zearalenone (ZEN)

Maize, barley, oat, rye, wheat, rice, Murphy et al. (2006), Lillard-Roberts sorghum, bread, corn, banana, sunower, (2004), Bennett and Klich (2003), walnut, cornake, sweetcorn Logrieco et al. (2003), Atalla et al. (2003), Hussein and Brasel (2001) Barley, maize, cheese, peanut, rice, peeled Pildain et al. (2008), Dombrink-Kurtzman barley, soybean, smoke-dreid meat, coffee (2007), Vinokurova et al. (2007), Murphy et al. (2006), Samson et al. beans, peanut, pistachio, millet seed (2006), Bennett and Klich (2003), Konietzny and Greiner (2003)

Cyclopiazonic acid (CPA)

Patulin

Barley, wheat, tomato, strawberry, banana, Bokhari et al. (2009), Dombrink-Kurtzman and Engberg (2006), Murphy et al. mango, avocado, apricot, blueberry, red raspberry, boysenberry juice, grape juice, (2006), Frisvad et al. (2004b), Bennett and Klich (2003), Konietzny and Greiner apple, pears (2003), Hasan (2000) Logrieco et al. (2009), Ostry (2008), Murphy et al. (2006), Lillard-Roberts (2004), Bennett and Klich (2003), Konietzny and Greiner (2003), Ren et al. (1998)

Alternariol monomethyl ether (AME)

Rice, olive, barley, oat, oilseed rape, Alternaria alternata, Alternaria sunower, maize, wheat, rye, mandarian brassicae, Alternaria capsici-annui, fruits, tomato, pepper, melon, Alternaria citri, Alternaria cucumerina, Alternaria dauci, Alternaria kikuchiana, Alternaria tomato, Alternaria solani, Alternaria longipes, Alternaria porri, Alternaria tenuissima Alternaria alternata, Alternaria brassicae, Alternaria tenuissima, Alternaria cucumerina, Alternaria capsici-annui, Alternaria citri, Alternaria dauci, Alternaria solani, Alternaria longipes, Alternaria porri Alternaria alternata, Alternaria tenuissima, Alternaria capsiciannui, Alternaria citri, A. cucumerina, Alternaria kikuchiana, Alternaria japonica, Alternaria longipes, Alternaria porri, Alternaria radicina, Alternaria tomato, Pyricularia oryzae, Phoma sorghina P. citrinum, P. expansum, P. radicicola, P. verrucosum, Monascus ruber, A. carneus, A. terreus, A. niveus Rice, sunower, mandarian fruits, olive, oat, barley, pepper, wheat, rye, oilseed rape, melon,

Alternariol (AOH)

Logrieco et al. (2009), Ostry (2008), Murphy et al. (2006), Lillard-Roberts (2004), Bennett and Klich (2003), Konietzny and Greiner (2003), Ren et al. (1998)

Tenuazonic acid

Rice, oilseed rape, olive, mandarian fruits, Logrieco et al. (2009), Ostry (2008), rye, barley, wheat, oat, pepper, sunower, Murphy et al. (2006), Bennett and Klich melon (2003), Konietzny and Greiner (2003)

Citrinin

Wheat, barely, rice, apple, medicinal plant seeds (Hydnocarpus laurifolia, Blepharis edulis, Piper betle, Acacia concinna, Caesalpinea digyna, Cassia stula, Argyreia speciosa, Embelia ribes)

Murphy et al. (2006), Frisvad et al. (2004b), (2005b), Anderson and Frisvad (2004), Overy and Frisvad (2003), Bennett and Klich (2003), Konietzny and Greiner (2003), Blanc et al. (1995)

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generate aatoxins (Frisvad et al. 2005a). The naturally occurring aatoxins designated as aatoxin B1, B2, G1, and G2. B and G forms are referred as they emit blue or green uorescence upon exposure to ultraviolet light (Murphy et al. 2006). These are the most toxic and carcinogenic secondary metabolites among the known mycotoxins (Ellis et al. 1991) and cause aatoxicosis upon ingestion of aatoxin in contaminated food or feed. Liver is the primary target organ for acute and chronic injury. Modied by-product of aatoxin B1 gets more toxic and carcinogenic during detoxication by the liver cytochrome P-450 monoxygenase. Their epoxide form binds to guanine residues inducing mutations (Yu et al. 2005). Since contamination of food and feed with mycotoxigenic fungi is a major problem, it is crucial to develop methods of detection for these pathogens that are relatively rapid and highly sensitive. Besides aatoxin-producing Aspergillus species, many species like A. fumigatus, A. avus, A. lentulus, A. niger, A. terreus, A. utus, A. nidulans act as opportunistic pathogens (Samson, 1994). Therefore, besides detecting them in human foods, it is also necessary to detect them in body uids for the diagnosis of aspergillosis and other Aspergillus-related diseases. Aspergillus species can cause several diseases in humans such as invasive aspergillosis, invasive pulmonary aspergillosis, allergic bronchopulmonary aspergillosis, and allergic fungal sinusitis (Segal 2009; Segal and Walsh 2006; Klont et al. 2004; Stevens et al. 2000; Schubert 2009). Early detection of these fungi can serve as a warning signal for the potential health hazard (Shapira et al. 1997; Varga 2006). Aspergillus species can cause several diseases in humans besides aspergillosis, such as aspergilloma, sinusitis, and allergic bronchopulmonary aspergillosis (Henry et al. 2000). The detection methods that are being used, such as cultivation on media and immunological (RIA, ELISA) methods, are time taking, laborious, and disadvantageous in some or the other way. However, for diagnosis of invasive aspergillosis, a commercialized ELISA-based method (Platelia test) is now commonly used for galactomannan detection in blood with improved sensitivity (0.51 ng ml-1) and specicity than the previous latex agglutination test with the same monoclonal antibody (Pinel et al. 2003; Giacchino et al. 2006). Chromatographic methods use costly and highly sophisticated equipments while serum-based methods work on one substance one assay concept (Konietzny and Greiner 2003; Yong and Cousin 2001). So, instead of detecting the toxins from aspergilli after its production, an alternative approach that can be used is to identify directly these molds before the toxin production by molecular methods. Due to the poor performance of immunological methods, scientists have started to diverge the attention toward the molecular diagnostic methods. Molecular methods are

generally based on detecting the differences in sequences of nucleic acids. Amplication, sequencing, DNA hybridization, and gel electrophoresis are some common laboratory methods that can be used for detection (Foong-Cunningham et al. 2006). Molecular methods such as PCR have facilitated the in vitro amplication of target sequences (Arnheim and Erlich 1992). The main advantage of PCR is that organisms can be identied within mixtures of DNA and without culturing the organisms. Thus, this method is both specic and fast. Probing, in combination with different molecular methods, has been found to be an effective way for the identication of specic amplicons in a mixture with similar sizes (Sandhu et al. 1995). A large number of probe-based methods have already been developed for the detection and enumeration of various foodborne pathogens (Jones 1991). DNA-based diagnostic methods have revolutionized the diagnostic technology in the clinical, forensic science, and in the agriculture sector. A number of DNA-based methods for the detection of Aspergillus are described in this review (Table 2).

PCR-based detections Several researchers have used the PCR method as probe to identify the specic organism or contamination on food and feeds. It is one of the easiest methods for the identication of any microorganism in samples. Manonmani et al. (2005) using an indigenously specic primer pair for the aatoxin regulatory (aR) gene assessed the presence of aatoxigenic fungi in foodstuffs. Although specicity was assayed with both pure and mixed cultures and only A. avus and A. parasiticus showed positive response of amplication, the limit of detection (LOD) of mycelium and spores was found to be 0.5 and C100 CFU, respectively. For A. avus, in groundnut and maize, the specicity and sensitivity was as little as 100 CFU g-1. Shapira et al. (1996), however, identied and sequenced three genes, versicolorin A dehydrogenase gene (ver-1), sterigmatocystin-o-methyltransfersase 1 gene (omt-1), and (an aatoxin biosynthesis regulatory gene) apa-2 from Aspergillus parasiticus. The primers only amplied A. avus and A. parasiticus genes while the amplication of other Aspergillus species, Penicillum species, and Fusarium species was not obtained. To assess the PCRs sensitivity, lowest level was amplied with ver-1 primers of A. parasiticus (102 spores g-1). Geisen (1996) and Farber et al. (1997) also developed a PCR system targeting norsolorinic acid reductase gene (nor-1), ver-1 and omt-1 genes. Both of them had discriminated the aatoxigenic Aspergillus species from the non-producers. However, the problem still stands with the A. avus group as some strains behave as non-aatoxigenic species. On the calmodulin gene basis,

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Arch Microbiol (2010) 192:409425 Table 2 Methods for detection of Aspergillus Detection method Polymerase chain reaction Specic primer of aR Calmodulin gene ver-1 gene 18S rRNA gene Calmodulin gene 18S rRNA gene 18S rRNA gene Hot start PCR Real-time PCR of mt tRNA & rRNA RTi-PCR of genomic DNA 0.5 CFU C100 CFU 12.5 pg 102 spores g-1 10 fg 10 pg 50 fg 10 fg 2 spores per reaction 0.2 GE 5 copies ml-1 SYBR Green I- 5 conidia per reaction TaqMan- 50 conidia per reaction Monochrome light cycler PCR Semi-nested PCR Nested PCR of ITS regions Multiplex PCR Combined methods LiPAPCR PCR-EIA PCR-EIA of 18S rRNA gene Nested-specic PCR-EIA of 18S rRNA gene PCR-EIA of mt gene DNA ngerprinting method RAPD DNA microarray ITS region of 18S rRNA gene Other molecular method SPC with immuno-uorescence labeling NASBA 210 hyphae per sample 1071011 copies ml-1 A. fumigatus A. fumigatus 10 pg Aspergillus, Candida Hsiao et al. (2005) A. carbonarius Fungaro et al. (2004) ITS50 pg ITS150 fg 0.5 pg 5 pg 1.7 ng ll-1 0.6 fg ml-1 Aspergillus Aspergillus A. fumigatus A. fumigatus Aspergillus Martin et al. (2000) 0.1 pg 0.01 pg 0.1 fg 10100 ag 110 cells A. avus A. fumigatus A. fumigatus, Rhizopus, Absidia A. fumigatus Aspergillus A. avus A. parasiticus A. carbonicus A. japonicus A. parasiticus A. fumigatus A. niger, A. tubingensis A. fumigatus, A. avus, A. niger, A. terreus, A. nidulans, C. albicans A. avus Aspergillus, Candida, Blastomyces, Histoplasma capsulatum, Sporothrix schenckii A. fumigatus, A. avus, A. niger, A. terreus A. carbonarius Sensitivity Target species References

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Manonmani et al. (2005) Perrone et al. (2004) Shapira et al. (1996) Bansod et al. (2008) Susca et al. (2007) Einsele et al. (1997) Zhou et al. (2000) Sandhu et al. (1995) Bolehovska et al. (2006) Selma et al. (2008)

Bu et al. (2005) Bialek et al. (2005) Zhao et al. (2001) Luo and Mitchell (2002)

Hinrikson et al. (2005) Elie et al. (1998) Golbang et al. (1999) Jones et al. (1998)

de Vos and Nelis (2003) Yoo et al. (2008)

Different methods used for detecting Aspergillus and other genus. (CFU Colony-forming unit, GE Genome equivalent, RTi-PCR quantitative Real-time polymerase chain reaction, ITS Intergenic transcribed spacer, LiPA-PCR Line probe assay polymerase chain reaction, PCR-EIA Polymerase chain reaction-enzyme immunoassay, RAPD Random amplied polymorphic DNA, SPC Solid phase cytometry, NASBA Nucleic acid sequence-based amplication)

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Susca et al. (2007b) identied Aspergillus niger and Aspergillus tubingensis, while Perrone et al. (2004) reported Aspergillus carbonicus and Aspergillus japonicus using PCR method. Susca et al. (2007b) developed a more sensitive detection for molds than Perrone et al. (2004) using the same gene and the same method. Li et al. (1998) identied the phylogenetic relationship between pathogenic species of A. fumigatus, A. avus, A. niger, A. terreus, and Emericella nidulans by PCR amplication of the mitochondrial cytochrome b gene. Except for Emericella nidulans, all other strains produced the 426-bp-long fragments. Species-specic nucleotides were found in each of the ve species. No difference between the strains was found after comparing the 142-amino acid sequences derived from the 426-bp nucleotide sequences. Kappe et al. (1998) identied pathogenic fungi (A. fumigatus, A. avus, Candida albicans, etc.) in human tissue with the help of amplication of 18S rRNA gene fragments. Three different types of oligonucleotide primers (TR1/CA1-TR2/AF2, UF1 and EU1) with an additional RZY1 primer were designed. The oligonucleotide AF hybridized with all known pathogenic aspergilli except A. niger and A. versicolor; oligonucleotide CA paired with three pathogenic Candida species. Similarly, targeting 18S region, Bansod et al. (2008) made a specic detection of Aspergillus fumigatus in patients with pulmonary tuberculosis by a two-step PCR. Again, Einsele et al. (1997) developed a PCR assay to identify fungal pathogens such as Aspergillus, Candida, Mucor, Penicillum, Trichosporon cutaneum, and T. glabrata. The species-specic primers and probes were designed by comparing the 18S rRNA gene sequences of these pathogens. The Southern blot detection system was 210 times more sensitive than the simple in-gel detection with ethidium bromide. The designed probes conrmed the species-specic hybridization and exhibited no cross-reactivity with others. However, the Aspergillus probe showed 100% identity with H. capsulatum; hybridization with this probe was additionally tested with probe specically hybridizing with A. fumigatus, A. avus, and A. versicolor, with positive results. Einsele and coworkers achieved this sensitivity, especially for Aspergillus species, by heating-alkaline denaturation-lysis for DNA extraction (DNA yield increased) and by amplifying a gene found in multicopy (C100 copy) numbers. Besides this, amplicon hybridization with labeled internal oligonucleotide and alkaline denaturation further improved the sensitivity of this assay. Sandhu et al. (1995) developed a highly specic oligonucleotide probe to identify the fungal infection of Aspergillus fumigatus, A. glaucus, A. niger, A. terreus, Candida, with Hot start PCR. Using 28S rRNA genes, 21 highly specic oligonucleotides were synthesized for approximately 50 fungal species. The specicity was determined by adding

50 pmol of probes, radiolabelled with [32P] ATP, on hybridization membrane under high stringency. The sensitivity of PCR primers was also evaluated and was found to be successfully amplied from as little as 0.2 genomes. In order to measure the air fungal concentration of 17 homes (indoor and outdoor) in Cincinnati, Meklin et al. (2007) did mold-specic quantitative PCR (MSQPCR) and showed that Aspergillus penicillioides followed by A. versicolor is present more than any other Aspergillus species. In another experiment, Zhou et al. (2000) detected ve other commonly found fungal species along with A. avus in indoor atmosphere and found that a minimum of two fungal spores was needed for the successful amplication by single primer within a time period of 56 h. Two modied semi-nested PCR assays for aspergillosis, caused by A. fumigatus, identication performed in parafn wax embedded tissue have been described by Bialek et al. (2005). Primers from 18S rRNA gene sequences were made and to raise the LOD, they introduced a third primer in the assay. A minimum amount of 0.1 fg of plasmid DNA (5 genome equivalent) was detected, assuming that there are 40 copies per genome. Cruzado et al. (2004) also tested two nested PCR methodologies on 27 strains of 16 genera and compared the results with previously reported results, for aspergillus detection. The detection limit obtained by the procedure given by Yamakami et al. (1996), differs by 500-fold in comparison with the method of Cruzado et al. who reported a limit of 65 fg instead of 110 fg which was reported by Williamson et al. (2000). The fungal pathogenic strain identication with nested PCR technique by amplifying the internal transcribed spacer (ITS) regions has been developed. This method specically detected A. fumigatus with sensitivity of 10100 attogram (ag) of sample DNA (Zhao et al. 2001). For speedy detection, multiplex PCR has been used to identify the pathogenic fungi directly from cultures. The fungi selected for the experiment were A. fumigatus, Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis. The detection was based on the species-specic primers designed from ITS regions of rRNA genes. The sensitivity of each set of primers ranged from 1001000 DNA molecules, representing the ability to amplify the appropriate amplicon from puried genomic DNA from 110 cells. The procedure was shortened by taking the 0.5 ll of fragments of hyphae, instead of extracting DNA (Luo and Mitchell 2002). Araujo et al. (2009) characterized A. fumigatus strains with microsatellite-based multiplex PCR and found that it is a simple technique to perform and shows high discriminatory power with excellent reproducibility (de Valk et al. 2008). Scherm et al. (2005) differentiated the aatoxin producers from non-aatoxin producers of A. avus and A. parasiticus strains, with the reverse transcription-polymerase chain reaction (RTPCR) technique. Total RNAs of

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13 strains were analyzed using specic primers based on the conserved regions of 9 structural genes (aD, aG, aH, aI, aJ, aK, aL, aM, aN, aO, aP, and aQ) and 2 regulatory genes aS and aR of the aatoxin B1 biosynthetic pathway. All these genes expression varied in response of aatoxin production and growth. Likewise, Degola et al. (2009) discriminated the aatoxin-producing A. avus (Alfa?) from the non-aatoxigenic (Aa-) through the same process; however, they classied some of the strain as slow aatoxin accumulators as these strains produced the mycotoxin after 10 days of growth. A few assays are successfully using the real-time PCR method like LightCyclerTM technique in combination with rapid in vitro amplication of DNA and detection (Buchheidt and Hummel 2005). Contamination problem and false results of PCR had made the real-time PCR assay, a suitable choice against the PCR methods. A well-designed assay, good primer choice, and probe sequence provide both sensitivity and specicity (Goebes et al. 2007). A large number of Aspergillus species as well as other genus have been detected from air and clinical samples using these methods. The threshold values have been reported between 500 pg and 50 fg. Real-time PCR (RTi-PCR) has also been used for the quantication of Aspergillus carbonarius in wine grapes. Genomic DNA from 52 fungal strains was extracted from the reference sample and food isolates to evaluate the specicity of the two primers and a probe. Comparatively, the LOD for SYBR Green I RTiPCR was found to be 10 times higher than the TaqManTM. Bangara et al. (2000) devised a SYBR Green quantitative PCR system for detecting aatoxin producer species of A. avus in black pepper based on the nor-1 gene sequence. The sensitivity showed by SYBR Green was 4.5 9 103 cells g-1 of pepper. In a similar way, Mayer et al. (2003) articially infected maize, pepper, and paprika to determine the minimal cell number for its infection by TaqManTM quantitative PCR. The detection limit for the PCR was little more sensitive (103 cells) than reported previously. In both cases, the infected commodities had shown a higher nor-1 gene copy number than the spore counts while the non-infected one had always negative result. Therefore, the RTi-PCR assay can be used for the prediction of probable toxigenic risk, even when there is very low levels of infection present (Selma et al. 2008). However, it had been suggested that high amount of plant genomic DNA interferes with the fungal DNA concentration and hinders the reaction. Therefore, careful DNA extraction is a crucial step that determines the sensitivity of the method. LightCycler real-time uorescence PCR, used by Imhof et al. (2003) and Bu et al. (2005), rapidly detected fungal infection in clinical tissue samples. Both species-specic hybridization assay and direct sequencing of amplication products identied A. fumigatus. Melting curve analysis of

Aspergillus species gave single products at 90C. Suanthie et al. (2009) while performing multiplexed real-time PCR for Aspergillus, Fusarium, and Penicillium species obtained a sensitivity of 1 pg to 10 ng in distillers grain (DG). They had designed pairs of broad-spectrum primers and probes from the ITS region of rDNA. In another experiment, Bolehovska et al. (2006) detected 103 clinical invasive aspergillosis samples out of 354 tested positive with this method. To identify the sensitivity of up to 5 copies mL-1, they had used two hybridization probes along with the primers. Schabereiter-Gurtner et al. (2007) proposed a novel real-time PCR assay for detecting 11 aspergilli and candida species in clinical samples. The analytical sensitivity varied according to the samples isolated. Sensitivity of pure culture was 1 CFU per PCR while that of blood- or CSF-isolated samples were 510 CFU mL-1 and 0.05 CFU lL-1, respectively. They had aligned the ITS2 regions of several pathogenic aspergilli species to obtain a consensus sequence for designing a single set of primer and increased the sensitivity using biotinylated probes. Species-specic detection of A. carbonarius and A. niger was done with real-time PCR by targeting the ITS, calmodulin, and polyketide synthase (pks) gene regions (Haugland et al. 2004; Mule et al. 2006; Auoti et al. 2007).

Combined methods For fast and sensitive determination of pathogenic fungal infections, several workers have combined two different techniques. The one is mostly PCR. Recently, singlestranded conformational polymorphisms (SSCP) together with PCR were used to screen the 11 Aspergillus species by the detection of calmodulin nucleotide variations (Samson et al. 2007a). SSCP indirectly detects a single base difference thereby affecting the strands mobility through a gel by altering the intra-strand base pairing and its resulting three-dimensional conformations (Susca et al. 2007c; Fujita and Silver 1994). For determining the medically important fungi from cultural or clinical samples, fungalspecic probes were designed by taking the ITS1 and ITS2 regions of 5.8S gene. All the tested strains Aspergillus, Candida, and Cryptococcus neoformans were unambiguously differentiated with the exception of cross-contamination from two Candida species. However, of all 21 specimens, the sensitivity was 100% where the speciesspecic probes were added in the PCR-reverse line blot (RLB) hybridization assay (Zeng et al. 2007). Martin et al. (2000) designed a method combining reverse hybridization line probe assay (LiPA) and PCR for the amplication of ITS regions of Aspergillus, Candida, and Cryptococcus. The assay is based on reverse hybridization principal.

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Specic probes are immobilized at identied sites on a nitro-cellulose membrane strip (Bosma et al. 2004) and are hybridized with the biotinylated PCR products of organisms DNA. The amalgams formed are consequently detected through colorimetric method (Rossau et al. 1997). This mingled method had 10-fold lower detection limit than the agarose gel electrophoresis for DNA. Cross-contamination results of specic probes with other pathogens were found in the experiment such as CD3 probe designed for C. albicans, paired with C. dubliniensis. However, with this assay, A. fumigatus can be detected in as low quantity as 50 fg ml-1 (1GE) and for C. albicans, threshold value was 210 cells ml-1 of blood. Polymerase chain reaction-enzyme immunoassay (PCREIA), a method that uses enzyme-bound antibody to detect the amplicons, was found to be specic and sensitive for A. avus, A. fumigatus, A. nidulans, A. niger, A. terreus, and A. versicolor identication (Hinrikson et al. 2005). PCR products were hybridized with digioxigenin-labeled probes on microtiter plate coated with streptavidin. Amplicons, designed from ITS1 and ITS2 regions, were detected on a single colorimetric enzyme immunoassay method. Thus, the aforementioned method proved to be ten times more sensitive than the conventional PCR detection. de Aguirre et al. (2004) had also found the same threshold value in their experiment with pathogenic fungal species. The benet of using PCR-EIA was that only small amount of DNA (in picogram) is needed for identication, DNA probes can reproducibly be synthesized and are very specic, probes can be stored ready for use, and the evaluation of results are instant (colorimetric and spectrophotometric measurements). Elie et al. (1998), however, had found the detection limit 10 times less than Hinrikson et al. (2005) for A. fumigatus 18S rDNA gene in a microtiter plate EIA. In another experiment, a nested-specic PCR-EIA, for A. fumigatus using 18S rDNA gene sequence, Jones with co-workers (1998) have got a much more sensitive method than that of Golbang et al. (1999). This may be because of the presence of high copy number of mitochondrial gene. Restriction fragments of different amplied regions (PCRRFLP) have shown a distinction between A. niger, A. tubingensis, A. carbonarius, and A. aculeatus isolates (Medina et al. 2005; Bau et al. 2006; Zanzotto et al. 2006; Martinez-Culebras and Ramson 2007). Similar results have also been obtained using species-specic primers in PCR for species level distinction. Targeting the calmodulin region, Perrone et al. (2004) and Susca et al. (2007a) differentiated A. carbonarius from A. niger while aiming the ITS regions, Haugland and Vesper (2002) detected A. carbonarius and A. niger and Gonzalez-Salgado et al. (2005) did species-specic identication for a number of black aspergilli. In diagnosing the invasive aspergillosis disease, Mirhendi et al. (2007) tested a single set of primer

and designed a restricted PCR prole for ve most pathogenic Aspergillus species. They also reviewed that both ITS regions are easy target areas of nucleotide sequence for discriminating the standard Aspergillus species.

DNA ngerprinting-based methods For typing of Aspergillus species, a number of molecular techniques have been developed recently. RFLP ngerprinting, MLPs, and MLST are being thought to have discriminatory power and reliability (Varga 2006; de Valk et al. 2008). Microsatellite length polymorphism (MLP), as the name suggests, microsatellites are amplied by PCR. These simple sequence repeats are present all over the DNA (Krackow and Konig 2008; de Valk et al. 2008). The simplest example of a microsatellite is a (CA)n repeats, where n is variable between alleles. These markers often present high levels of inter- and intra-specic polymorphism, particularly when tandem repeats number are ten or greater (Queller et al. 1993). Multilocus sequence typing (MLST) is a technique in molecular biology for the typing of multiple loci. MLST involves sequencing of internal fragments of multiple (usually seven) housekeeping genes. MLST directly measures the DNA sequence variations in a set of housekeeping genes and characterizes strains by their unique allelic proles. The principle of MLST is simple: the technique involves PCR amplication followed by DNA sequencing. Nucleotide differences between strains can be checked at a variable number of genes (generally seven) depending on the degree of discrimination desired (Maiden et al. 1998). Several workers had performed RFLP of mtDNA, rDNA, pectin lyase (pelA), and pyruvate kinase (pki) genes to distinguish between different black aspergilli species (Varga et al. 1993; Varga et al. 1999; Parenicova et al. 2001; de Vries et al. 2005). Likewise, Perrone et al. (2006) have analyzed the polymorphism of amplied fragments so as to distinguish all the black aspergilli species at molecular level. However, to estimate the level of aatoxins in the Aspergillus avus, Baird et al. (2006) used DNA amplication ngerprinting (DAF) method to determine regions of ITS1 and ITS2. Aspergillus carbonarius was identied using an RAPD-based method (Fungaro et al. 2004). A random gene fragment was selected whose specic oligonucleotides primers were synthesized and used for amplications. An 809-bp fragment was detected only from the A. carbonarius DNA. Once this marker was sequenced, it was transformed into a unique and robust PCR-based marker. With the experiment, it was found that there exists no relationship between RAPD genotyping and mycotoxigenic properties of strains. When compared to conventional method, results were obtained within 24 h of time. Comparing the three DNA ngerprinting methods

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(RFLP, MLP, and RAPD), Bart-Delabesse et al. (2001) found that out of total 67 isolates of A. fumigatus tested, 49 genotypes with 37 unique types were differentiated by RFLP, while MLP analysis yielded 28 unique patterns in 43 distinct genotypes whereas RAPD showed only 31 types with 17 unique types. Therefore, the polymorphisms were in order of RFLP [ MLP [ RAPD. The MLP and RFLP methods are based on specic DNA hybridization between either a probe or primer and target DNA. Thereby, they have higher reproducibility than that of the RAPD. RFLP of mitochondrial DNA of Aspergillus section Flavi has been used for their taxonomic differentiation. Mitochondrial DNA restricted with HaeIII, AreI, or DraI gave unique band with each aspergillus species; however, with some restriction enzyme(s), similarity patterns may arise in bands (Quirk and Kupinski 2002). In another study, amplied fragment length polymorphism (AFLP) and genomic DNA sequencing were used to identify 77 black aspergilli strains from grapes. It was observed that they shared \25% of AFLP similarity and belonged to four main distinct divisions were identied. Perrone et al. (2006) suggested that AFLP could be utilized as a tool for studying genetic diversity of Aspergillus species. Thus, with the help of the AFLP method, genetic relatedness and/or resolving relationships within or in between two closely related groups or species could be evaluated. McAlpin and Mannarelli (1995) designed a DNA probe for differentiating A. avus strains. The repetitive DNA sequences have proven to be a useful and reliable character in evaluating genetic relatedness of strains at different levels of taxonomic classication, and the probe was created by DNA ngerprinting technique. The 6.5-kb pAF28 DNA probe hybridized with DNA of closely related varieties including A. avus var. oryzae, A. avus var. parasiticus, A. avus var. sojae, and A. nomius. However, no hybridization was observed when the probe was hybridized with the other fungal species DNA such as A. ochraceus NRRL402, A. auricomus NRRL391, A. alliaceus NRRL315, F. moniliforme NRRLA-28160, and P. thomii NRRL6218.

DNA microarray-based methods Microarray has rapidly become one of the standard laboratory tools for identication as well as quantication of many specic DNA sequences in complex nucleic acid samples (Chen et al. 1997; Jayapal and Melendez 2006). Microarray-based proling is a powerful approach for the diagnosis of disease targets. The array detection for gene expression can serve both as markers in addition of detection of expression level (Jayapal and Melendez 2006).

For the rst time, Leinberger et al. (2005) applied DNA microarray technology for the recognition and identication of fungal mycoses. Twelve Aspergillus and Candida species, which most frequently cause this disease, were targeted. For detection, 1624 bases of oligonucleotide probes were designed by exploiting the conserved nature of ITS1 and 2, thereby, making these probes as genus specic. Although the cross-hybridization had been observed in Candida species which can be resolved by designing additional species-specic probe, yet this method have been found to be better than any other technique. The whole procedure takes only 4 h for determination of 12 species, after DNA extraction. In a similar way, Hsiao et al. (2005) also identied 228 pathogenic fungi. ITS regions of 18S rRNA genes were PCR-amplied, and products were used for designing the probes. The uorescently labeled probes were then immobilized on the nylon membrane of a microtiter plate. With this DNA chip technology, 64 clinically important fungal species of 32 genera were determined. From isolating the colonies to the testing of array, the whole procedure was nished in 24 h, with a detection limit of fungal genomic DNA as low as 10 pg. To identify fungal pathogens in neutropenic patients, an assay of multiplex PCR in combination with DNA microarray hybridization was used by Spiess et al. (2007). Conserved regions of 18S, 5.8S, and ITS1 of rRNA genes were used for PCR primer designing and capture probes. Hybridizing the fungal genomic DNA with capture probes resulted in the species-specic identication of A. fumigatus, A. avus, A. terreus, and other pathogenic fungal species. Schmidt-Heydt and Geisen (2007) used the same microarray technology in order to monitor the mycotoxin production in food. The basis of microarray was to detect the activation of all gene clusters under favorable conditions and to add newly identied pathway genes, at any time. The resulting signals were specic under hybridization conditions. Schultz et al. (2008) showed a novel uorescent biosensor which detects 16 molecules lm2 which nally can be reduced to 1 mol lm2 if the system is conned to photon noise. However, Laitala et al. (2007) used decay time, provided by decay probe (europium as donor and organic uorophore as acceptor), to discriminate hybridized probe population from unhybridized population. In a similar way, for fungal identication, Wang et al. (2008) also obtained uorescent signals from as low as 0.5 nM of oligonucleotide signals and detected 3 ng of amplied product. For detection of black aspergilli (A. carbonarius, A. ibericus, and A. japonicus) on grapes, Bufier et al. (2007) and Susca et al. (2007a) manufactured a new type of lowcomplexity oligonucleotide microarray: OLISATM OLIgo Sorbent Arrays. This biochip consists of a series of 16 probes at each well of a microtiter plate. The specimen is

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PCR-amplied with biotin-labeled PCR primers, denatured, and then specically hybridized to any single nucleotide polymorphisms of an amplied DNA target. The spot pattern allows the automated identication of the species present in specimen. Identication of multiple species of Aspergillus simultaneously can be achieved by combining multiple amplication and detection method. Chip assays have proved their superiority by requiring small amount of amplied products and generating huge amount of data, and its analysis is also rapid and can be automated. Array systems have been designed for the use in biomedical analysis, environment monitoring, or bioterror agents detection. The basic purposes of these microarray biosensors are portability, automatic recognition, robustness, and cost-effectiveness. The cost of these microarray chips can be reduced to minimum level if glass slides are used for its manufacturing.

Other potential molecular methods With solid phase cytometry (SPC) in combination with immunouorescence labeling, distinction between Aspergillus species and other pathogenic fungi can be made within an hour (de Vos and Nelis 2003). The same technique was used by de Vos et al. (2006) with addition of laser scanning for the identication of Aspergillus fumigatus hyphae in respiratory secretions. Depending upon the enzymatic viability staining, with carboxyuorescein diacetate, specic and non-specic detections of fungal hyphae were performed. Specic detection needs preincubation at 45C with viability staining whereas for nonspecic identication, only staining procedure is required. As low as 210 A. fumigatus hyphae per sample have been diagnosed in spiked sputum using non-specic and specic staining in 2.5 and 8.5 h, respectively. Yoo et al. (2008) used quantitative real-time (RTi) nucleic acid sequencebased amplication (NASBA) for identifying Aspergillus fumigatus. They constructed an internal control (IC) RNA from Calvibacter michiganensis subsp. sepedonicus strains. Internal control RNA contained a 6-carboxy-Xrhodamine (6-ROX) uorescent dye whereas A. fumigatus beacon was labeled with 6-carboxyuorescien (6-FAM) at its 50 -end. Internal control RNA was detectable in the ranges of 1071011 copies ml-1, which helped in predicting the unknown concentrations of target RNA using titration curve. This method showed the sensitivity of 96% of identication of A. fumigatus. NASBA-LF was 10 times more sensitive than competitive-PCR and 1000 times more than RTPCR. Its result had led the authors to consider the test as gold standard with 100% specicity and sensitivity (Olmos et al. 2007). To detect intraspecic variations of aspergillus group, Kumeda and Asao (2001) developed a novel heteroduplex panel analysis (HPA) using PCR-amplied regions of ITS. This method involves a panel heteroduplex formation which was compared with a set of standard species-specic panel. This typing discloses its discriminatory power by detecting even a single base pair difference within species. Because of the high discriminatory power and high throughput, short tandem repeats (STRs) has been increasingly used as genetic markers. Their overall presence in most higher organisms and polymorphisms have made them important for strain identication and discrimination in a wide variety of microorganisms. Ciardo et al. (2007) proposed to develop an algorithm for the identication of various molds on the basis of molecular and phenotypic methods. They have created an IMM database covering almost all the medically signicant molds based on ITS regions.

Methods based on retrotransposon insertion-site context (RSIC) typing de Ruiter et al. (2007) used a novel type of PCR alternative which is less time-consuming and easier than the RFLP or Southern blot analysis. It has been shown earlier that retrotransposons offer very stable ngerprints. Therefore, they can be employed for typing-like assays. It is a hemi-nested ligation-dependent sort of PCR assay. RSIC utilizes the occurrence of a multicopy element dispersed in the whole genome and indents to amplify the anking sequences of retrotransposon elements. Retrotransposons were characterized by the presence of two LTRs encircling the three coding regions. After the genomic DNA isolation, a combined restriction-ligation procedure was used to join the adapters. The restriction-ligation was PCR-amplied, and then the amplicons were analyzed. The data obtained were further studied with bioinformatic softwares. On analyzing the 55 A. fumigatus samples from 15 patients, 20 bands were obtained with a range of 60300 bp. This means that they belonged to same genotype. Comparatively, Aspergillus, isolated from respiratory samples, had shown different genotyping within the individual patients. The major difference between the AFLP and RSIC and STRAf (Short tandem repeat with reference to Aspergillus fumigatus is known as STRAf) is that the later ones are much more sensitive than the AFLP. RSIC and STRAf can determine two different genotypes from two patients while AFLP cannot distinguish the difference between them (de Ruiter et al. 2007; Klaassen and Osherov 2007). Retrotransposons are also present in other aspergilli species such as Aspergillus avus and Aspergillus nidulans.

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Future aspects In recent years, numerous additional novel ways have emerged to improve the protocols for diagnosis of the pathogens and to overcome the drawbacks. Sophisticated molecular techniques are being developed that may be used to screen the particular fungus within a range of population. But before performing the molecular detections, it is imperative to extract the nucleic acid from the samples. For this, the samples may be divided into two categories: (1) medical sample material such as blood or bronchoalveolar lavage (BAL) samples (2) contaminated agricultural food and feed samples as peanut, wheat, cereals. For medical samples, no universal method has yet been obtained for the source of sample collection and nucleic acid extraction protocol. However, Loefer et al. (2000) suggested the whole blood in EDTA (Klingspor and Loefer 2009)containing tubes could be a better sample for fungal DNA isolation purpose. The advantage of this sample is that both free- and the cell-associated DNA can be easily assessed, EDTA in the tubes inhibit DNases activity present freely in the blood without interfering the PCR assay (Garcia et al. 2002). DNA isolation is, currently, seen as the target of choice due to its relative stability and ease of extraction against RNA; still the extraction methods provide variations in DNA concentration. In the similar way, for contaminated plant materials, no universal method is present for DNA isolation. However, the type of sample collection is not a problem here as one can take only the contaminated part of the plant. The major problem is the PCR inhibitors present in the plant material itself. Besides this, the quantity of fungal concentration on the contaminated material is also a problem. To eradicate this problem or to increase the quantity, enrichment procedures are used. But that needs 35 days of time causing further delay in detection. Therefore, a single and consensus method should be gained for extraction of DNA for both medical as well agricultural materials which does not require enrichment procedure (Table 3). In a recent research, Balajee et al. (2007) and Samson et al. (2007b) recommended the use of ITS regions as a convenient universal marker for fungal species identication. Microsatellites or STRs under all altered conditions produced a detectable signal. Thus, the STR assay had proved to be an extremely robust method (de Valk et al. 2007; Klaassen and Osherov 2007) to determine the presence of Aspergillus species. Identication and typing of Aspergillus from cultures or environmental samples have resulted in development of more and more PCR-related technologies or other molecular approaches. However, a molecular gold standard assay is yet to be produced (Buchheidt and Hummel 2005). The assurance of PCR and related methods to identify

Aspergillus has subsided the potential disadvantages. As suggested by Niessen (2007), the new assays should be rst checked through in silico method for the cross-reactivity and the feasibility of the reaction to obtain good results. Contamination in the PCR mixtures, interference of related fungi with the target fungus (Klingspor and Loefer 2009), shared genes for different mycotoxins (sterigmatocystin and aatoxin), disrupted gene presence without the production of toxin are few problems that may provide false results. In addition, dead cells or pieces of DNA may also provide results. Thus, methods are to be devised that may provide on-site detection and are cost effective and easier to use. DNA microarray and probe technology can be quite useful to make the aatoxigenic fungus estimation ready-to-use, cost effective, and less time-consuming procedure. Few recently developed methods can be taken into consideration for this purpose and can be modied accordingly (Table 3). Varallyay et al. (2007) utilized LNA (Locked nucleic acid) probes for estimating microRNAs (miRNAs). This method allows sensitive and highly specic detection of mature miRNAs. Circularized oligonucleotide probes (C-probe) have proved to be a considerable advancement in the area of nucleic acid detection (Zhang et al. 2006). Several authors have reported the real-time technology of RCA and RAM (Faruqi et al. 2004; Nilsson et al. 2002; Tyagi and Kramer 1996). Inversion probes along with pyrosequencing for detecting Mycobacterium tuberculosis can also be used for fungal identication, as it is a very sensitive technique. The detection limit for M. tuberculosis DNA was found to be 500 fg (Novais et al. 2008). Besides direct detection of ssDNA or dsDNA, the technique is also more accurate and much faster than culture-based method. Sequence-enabled reassembly with green uorescent protein (GFP), b-lactamase (LAC), or mCpG can also be utilized directly to quantify the dsDNA (Ghosh et al. 2006). In an attempt to estimate the fungal pathogenic nucleic acids, Wang et al. (2008) devised a microuidic microarray kit. Compared to the PCR, which detects 3 ng of DNA, this device provides uorescent signals from as small as 0.5 nM (1 lM) of DNA. Dore et al. (2006) reported an ultra sensitive and sequence-specic DNA detection system. They had shown that a suitable uorophore with kexc = 530 nm and kem = 575 nm increases the sensitivity dramatically, allowing the detection limit in zeptomolar (10-21) concentrations in just only 5 min without any prior target amplication. These newer techniques can be used for the Aspergillus detection, which will further enhance the sensitivity of identication. Though recent techniques are quite good and efcient yet they need to be modied for the sake of

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420 Table 3 Future applications Technique name MIP ? Pyrosequencing Working Pyrosequencing is a real-time DNA sequencing. Instead of simple primers, MIPs (circularized ss-oligonucleotides) are used. With amplication, PPi is released turning AMP in ATP by ATP sulfurylase enzyme. The ATP provides energy to luciferase for oxidizing luciferin to generate light. This light is quantied by pyrosequencer It is a type of DNA sensor based on electrostatic interactions between positively charged optical transducer and uorescently labeled negative DNA probe. Transducer gets quenched after joining with ssDNA probe which then illuminates when a complementary oligonucleotide sequence is added to it Amplication starts by DNA polymerase extends single forward primer bound to C-probe through RCA. RAM utilizes two primers. Forward primer is same as RCA to generate ssDNA which the reverse primer, having identical sequence to C-probe, uses for secondround extension. In Northern blot assay, LNA probes are used to increase the sensitivity by enhancing the hybridization capacity. Modied LNA probe detection is sensitive to miRNA by about 10folds than regular DNA The amplied PCR product is used for generating the DNA probes that are to be used with ISH. Bearham et al. (2008) tested the DNA probes for detection of Minchinia sp. from rock oysters It is a type of probe amplication where specic synthetic ssDNA (RP) joins the template. The restriction nicking endonuclease cuts the recognized site resulting in the reduction in Tm and subsequently releasing two smaller ssDNA probes. The same RP is used to cleave again and again generating large amount of ssDNA copies. This method can be used for both DNA and RNA. It can specically detect samples within 515 min It is an isothermal amplication method for RNA basically. First primer attaches to the complementary end of 3 template to synthesize cDNA by reverse transcriptase. Second primer attaches to 5 end of DNA and T7 RNA polymerase again synthezises cRNA. With lateral ow-through, enhanced binding rate reduces hybridization time Detection limit 500 fg

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References Novais et al. (2008)

Signal detection of ss/ds DNA

3 zM

Dore et al. (2006)

Amplication by C- probe

Zhang et al. (2006)

Detection through LNA probe

Varallyay et al. (2007)

Detection by DNA probes

10 fg of template DNA in 250 ng of host DNA

Bearham et al. (2008)

RIDA

Gao et al. (2008)

NASBA-LF

2 copies of DNA

Olmos et al. (2007)

(MIP molecular inversion probe, zM zeptoMolar (10-21M), C-probe circularized probe, RCA rolling circle amplication, RAM ramication mechanism, LNA probe locked nucleic acid probe, miRNA microRNA, ISH in situ hybridization, RIDA rapid isothermal detection assay, RP reporting probe, NASBA-LF nucleic acid sequence-based amplication-lateral ow-through)

identication of fungal genomes and need to be further improved before they are used for diagnosis purposes. Further, a thorough study of all human pathogenic

Aspergillus species is needed to improve the understanding about the virulence and variations present in the natural isolates.

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Arch Microbiol (2010) 192:409425 Acknowledgments SJ is thankful to Department of Science and Technology, Government of India for nancial assistance.

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