Study on the internal control on polymerase chain reaction in Yersinia pestis detection / 中华流行病学杂志

ABSTRACT

<p><b>OBJECTIVE</b>For the detection of Yersinia pestis by polymerase chain reaction (PCR), internal control (IC) is required in order to prevent false negative results that might be caused by PCR inhibitors.</p><p><b>METHODS</b>F1 antigen was amplified by PCR with primer F1 and the PCR product of primer F1 were cloned with TOPO TA cloning Kit. The plasmid of positive clone was then digested with HpaI. The digested plasmid and the PCR products of 16S rRNA were ligated with T(4) DNA ligase before the ligated products were transformed. Isolate plasmid DNA on positive clone and its concentration were measured. Plasmid DNA on different concentration by PCR amplification with primer F1 was analyzed and the standard concentration of IC was determined.</p><p><b>RESULTS</b>Constructing an IC by inserting a 16S rRNA amplicon to the original target DNA between the two primer F1 sites, the size was longer than the target DNA. The standard concentration of IC was determined.</p><p><b>CONCLUSION</b>An optimal IC concentration to increase the reliability of the PCR assays might be used to prevent false negative results and appeared to be useful for detection of Yersinia pestis.</p>