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CONTENTS
INTRODUCTION AIM AND OBJECTIVE METHODOLOGY RESULTS DISCUSSION
INTRODUCTION
Malaria , a disease that has caused untold misery throughout the world since antiquity. Caused by Plasmodium (vivax , falciparum , ovale, malariae )
Sporozoites
Derived from Greek word ( sporo = seed &
zoon = animal) Shape : spindle-shaped, elongated. Size: 11 m in length and 1 m in diameter The infectious stage of the Plasmodium life cycle, the form in which malaria is passed from the mosquito vector to the mammalian host . Circulate through the body and invade liver in a short time. Despite their short persistence in circulating blood, induce a strong immune response
o o o o The species of Anopheles involved in the transmission of o human malaria world-wide are o incredibly diverse . o o There are 58 species of Indian o anophelines in different o ecological settings in India o Salivary glands of female Anopheles mosquitoes. A. culcifacies A. fluviatilis A. stephensi A. dirus A. minimus A. sundaicus A. annularis A. varuna A.jeyporiensis A. philippinensis A. Subpictus*
Methodology
To perform circumsporozoite(CS) protein ELISA To isolate DNA from Anopheles mosquito To identify the species of Anopheles by PCR Analysis of PCR products by agarose gel electrophoresis. Sequencing of PCR product
CIRCUMSPOROZOITE ELISA
As malaria sporozoites posses a major surface Ag , the CS protein which uniformly surrounds their external coat , that is infective to the vertebrate host. MAbs raised against this protein are used in the field to detect which species of Anopheles vectors are involved in malaria transmission. The most attractive alternative to microscopy is CS ELISA sandwich assay. Considered to be the gold standard
CS ELISA
(1) Adsorption of capture MAb to the wells of a microtiter plate. (6) Test samples are then added to the wells (7) Well contents aspirated & washed twice with PBST:20
(5)
(4) Each well was then completely filled with blocking buffer
(9) Well contents were aspirated & washed 3-4 times with PBST -20
(10)
(11) (12)
supernatant removed
Air dried
Taq buffer
Mgcl2 DNTPs
1mM 160 M
200 nM
200 nM 1 U /reaction
50-200 ng
95 C 55 C 72 C 4 C
Number of cycles = 35 cycles 1.Forward Primer D3A ( 5 GAC CCG TCT TGA AAC ACG GA 3) 2.Reverse Primer D3B (5 TCG GAA GGA ACC AGC TAC TA 3 )
The slurry collected in the extraction column and eluted DNA in collection tube
Sequencing PCR
Ingredients 1. Reaction reagent 1X 0.5 14X (required volume in l) 7
2 1.75 5.75 10
28 24.5 192.5 2
96 50 60 4
Washing was done with 70% ethanol and spin at 12000 rpm for 10 min.
Hi-Di formide (12l)was added. Tubes were snapchilled & proceeded for sequencing
RESULTS
Isolation of DNA :-
228 A. fluviatilis
307 A.culcifacies
535 mosquito
Figure :- showing amplification of isolated DNA samples from both A.fluvitalis & A .culcifacies Lane 1 -5 = shows isolated DNA samples from A. fluvitalis (375 bp product) Lane 6 = 100 base pair ladder. Lane 7 11 = shows isolated DNA samples from A. culcifacies (372 bp product)
Detection of Plasmodium sporozoite of human origin in mosquito salivary glands is important to determine which species of Anopheles is more prevalent in a particular region. As the correct identification of any vector implicated in malaria transmission is key to successful control. Failure to recognise sps of Anopheles may result in failure to distinguish between a vector & a non-vector. Hence the assessment of the impact of control measures may be seriously misleading.