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A TaqMan Fluorescent Quantitative Polymerase Chain Reaction Assay for Pathogenic Leptospira Spp.

Author: LiChunJuan
Tutor: ZhangYiBing
School: Liaoning Medical
Course: Clinical Laboratory Science
Keywords: Leptospira spp. detection and identification lipL32 gene TaqMan probe
CLC: R440
Type: Master's thesis
Year: 2011
Downloads: 22
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Abstract


ObjectiveTo establish a highly sensitive, specific, stable and rapid Fluorescent Quantitative Polymerase Chain Reaction method for detection of Leptospira spp with SmartCycler, and certify using LightCycler 2.0 and ABI7300 instruments.MethodsBased on the outer-membrane lipoprotein L32 gene sequence of strain CMCC 56601 of Leptospira spp recorded in the GenBank, two pairs of primer and one TaqMan probe were designed. Specific gene fragments obtained by PCR were cloned into PMD18-T vector to construct recombinant plasimdse, which were then linearized with restriction enzyme BamH I and were identified their specificity by direct PCR and DNA sequencing. We used the TaqMan probes and SmartCycler of Cepheid to develop FQ-PCR assays for the rapid detection of Leptospira spp. FQ-PCR reaction system was optimized to determine the optimal concentrations of primers and probes. The sensitivity and specificity of FQ-PCR was evaluated. we investigated whether these assays produce comparable specificifiy and sensitivity using different equipments including SmartCycler, LightCycler2.0 and ABI7300.ResultsThe specificity of FQ-PCR was evaluated using 35 strains. In conclusion, the FQ-PCR unequivocally distinguished target strains from nontarget bacteria. Using prior centrifugation, The sensitivity of the FQ-PCR assay for linear recombinant plasmids DNA was 10 copies.The sensitivity determined using 10-fold dilutions of purified gDNA was 10 fg per reaction for FQ-PCR assay. The assay was able to quantifiably detect 2×10 cfu/ml, for Leptospira CMCC 56601. It is capable of detecting 2.0×102 cfu/ml for contaminated water sample. The slopes of the linear regression curves calculated over a 7/6-log range were similar to the theoretical optimum value of -3.32, which showed amplification to be very efficient. The quantification was linear (R2≥0.995 with a PCR efficiency of≥90%). This FQ-PCR method was established using the Smartcycler, LightCycler2.0, ABI7300, the similar results were available in these different FQ-PCR instruments.ConclusionsThese FQ-PCR assays for the detection of Leptospira spp. were developed, which were rapid, sensitive, specific, and can be used on different types of instruments, with a wide range of applications in public safety, health monitoring, epidemiological investigation, clinical diagnosis and so on.

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