Improvement of the caf1 based PCR method for detection of the plague / 中华地方病学杂志

ABSTRACT

Objective To identify the causes of nonspecific bands in the detection of a industry standard caf1 gene by polymerase chain reaction(PCR),and to propose a solution to this problem. Methods A total of 112 strains were selected for the experiment, including 40 strains of Yersinia pestis, 72 strains of non-Yersinia pestis;DNA was extracted,and caf1 gene was amplified by PCR;seven non-specific strips were recovered,purified and TA cloning and sequencing; the primer of the caf1 gene was redesigned and validated using all of the strains. Results Using the industry standard caf1 gene primer,DNAs of 40 Yersinia pestis and 72 non-Yersinia pestis were amplified by PCR, 58 non-Yersinia pestis could be amplified with non-specific bands, they were about 400, 500, 600, 700, 800, 900, 1 000 bp. By TA cloning and sequencing, the non-specific bands in the downstream of the industry standard caf1 primer and its reverse complement were amplified. Using the new designed caf1 primer to amplify, 72 non-Yersinia pestis strains showed no non-specific bands. Conclusion Non-specific bands has been amplified in the screening of Yersinia pestis using the primer of the industry standard caf1, and the new caf1 primer can effectively avoid this problem and improve the accuracy of detection.