Development and application of TaqMan MGB probe fluorescence quantitative PCR method for rapid detection of Clostridium piliforme / 中华流行病学杂志

ABSTRACT

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.