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[Development and application of TaqMan MGB probe fluorescence quantitative PCR method for rapid detection of
Zheng-qin Gao1, Bing-fei Yue, Zheng-ming He
1National Institutes for Food and Drug Control of China, Beijing, China. gaozhengqin@126.com
Objective:
To develop a TaqMan MGB probe-based, sensitive and specific fluorescence quantitative PCR assay method for rapid detection of Clostridium piliforme.
Methods:
Primers and probes specific to 16S rRNA gene of Clostridium piliforme were designed. A TaqMan MGB probe-based, fluorescence quantitative PCR method was established. Specificity, sensitivity and stability of the method were assessed, followed by real-time quantitative PCR assay to detect Clostridium piliforme on 1156 clinical specimens during 2008-2011 and compared with conventional PCR assay.
Results:
The specificity of TaqMan MGB probe-based fluorescence quantitative PCR was high and did not show cross-reactivity with Helicobacter hepaticus, Helicobacter pylori, Campylobacter jejuni, Pasteurella pneumotropica, Escherichia coli or Pseudomonas aeruginosa. The detection limit was 2.2 copies/µl. The correlation coefficient and slope value of standard curve were 0.999 and -3.204, respectively and the efficiency of TaqMan MGB-based probe fluorescence quantitative PCR assay was 100%. When the TaqMan MGB-based probe fluorescence quantitative PCR assay was preformed to detect Clostridium piliforme on 1156 clinical specimens, a total of 101 specimens showed positive on Clostridium piliforme. However, only 44 specimens showed positive when conventional PCR was used. The real-time quantitative PCR for Clostridium piliforme could be completed within 2 hours.
Conclusion:
The TaqMan MGB-based probe fluorescence quantitative PCR assay method was a reliable, specific, sensitive and useful tool for rapid detection of Clostridium piliforme.
