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Summer Research Fellowship Programme of India's Science Academies

Isolation of DNA and PCR amplification of Vitamin-D Receptor (VDR) gene from patients of Post Kala-Azar Dermal Leishmaniasis (PKDL) in Bihar

Manoj Kumar Sharma

Janta Vedic College, Baraut, Baghpat 250611 (CCS University, Meerut) Uttar Pradesh

Dr.Vahab Ali

Scientist-E, Department of Biochemistry, ICMR-Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna 800007

Abstract

Leishmaniasis, the life-threatening disease caused by the digenetic, obligateintracellular, protozoan parasites of genus Leishmania is transmitted by sand flies and is prevalent mainly in poor socio-economic and tropical regions. In India, Visceral leishmaniasis (VL or Kala-Azar), the fatal disease form causing severe Hepato-splenomegaly, has significantly been controlled by on-going Kala-Azar Elimination Programme implemented since last two decades; but the dermal sequel of VL i.e. Post Kala-Azar Dermal leishmaniasis (PKDL) is more observed, probably due to the parasitic transmission dynamics. Etiologically, drug resistance/non-responsiveness, immune-suppression in persons along with other environmental factors such as high vector density, UV radiation (UVR) exposure etc. are mainly responsible for PKDL infections; still there are scientific reports indicating some significant role of Vitamin-D receptor (VDR) gene polymorphisms in the pathogenesis of PKDL/VL endemic population. UVR helps in synthesis of the essential Vitamin-D in human skin, whose deficiency is associated with aetiology of VL/PKDL in patients. After taking the Questionnaire study, as per the Ethical Committee (EC) protocol, the blood samples of 21 PKDL, 4 VL patients and 3 healthy persons were collected at ICMR-RMRIMS, Patna and their genomic DNA was isolated by Qiagen kit. The extracted DNA samples were electrophoresed in Agarose gel (0.8%) and single band of isolated DNA, each was resulted approximatly 21kb. The amounts of DNA from 400μl blood samples were about 10-30 μg. The quantity and quality of isolated DNA were confirmed by UV-VIS double beam-(Hitachi) and Nano drop-(Genetix) Spectrophotometry, where the absorbance readings at 260 nm and 280 nm were taken. Lastly, the PCR amplification of VDR genes (Bsm1 and Taq1) was performed to study the role of VDR gene polymorphism in the pathogenesis of PKDL in Bihar with help of RFLP or DNA sequencing in future.

Keywords or phrases: PKDL pathogenesis, Vitamin-D, PCR product, VDR gene polymorphism.

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