Comparison of DNA extraction methods and gene targets from Leptospira interrogans serovar icterohaemorrhagiae for application in polymerase chain reaction
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Authors
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S ANITHA
Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu 600007 India
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G DHINAKAR RAJ
Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu 600007 India
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A RAJA
Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu 600007 India
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A THANGAVELU
Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu 600007 India
Keywords:
Analytical sensitivity, DNA extraction methods, Gene targets, Leptospira, Polymerase chain reactionAbstract
The objective of this study was to identify the appropriate DNA extraction method and gene target from Leptospira interrogans serovar icterohaemorrhagiae for subsequent application in polymerase chain reaction (PCR) for diagnosis. DNA was extracted from pure cultures of Leptospira interrogans serovar icterohaemorrhagiae, spiked serum and urine samples using 4 different protocols,viz. STET buffer method, boiling method, nucleospin kit and the QIAamp DNA extraction kit using LipL41, LigA/B and LipL32 genes as targets. The lowest concentration of DNA that showed a visible band in PCR was taken as its analytical sensitivity in terms of concentration of DNA for each type of sample, target gene and DNA extraction method respectively. It was observed that DNA extraction from suspected clinical human sera samples using the QIAamp kit with either LipL41 or LipL32 as targets were most suitable for diagnosis by PCR since it had the greatest sensitivity of detection in PCR and the lowest amount of contaminating protein in the extracted DNA.
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