RESUMEN
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.
RESUMEN
Objective To get recombinant F1 antigen (rF1) and to construct the detection dipstick of plague antibody. Methods The cafl gene removing the signal peptide coding sequence was cloned into plasmid pET32a ( +) by double-digested sites of BamHI and Not I. Recombinant plasmid caf1-pET32a(+) was transformed into BL21 (DE3) and the rFl was expressed. Expression products were purified by affinity chromatography. Dual detection dipstick of plague antibody was constructed with purified rF1 and natural F1, and evaluated with 528 human serum samples of Zhejiang province. Results The fusion protein rF1 of 35.5 KD was expressed by BL21 strains containing caf1-pET32a( + ). The sensitivity of rF1 showed equivalent to or higher than the natural Fl antigen in detecting plague antibody. It seemed that there was a better consistency of 97.9% (k= 0.466) when 528 human sera was detected by rF1 and natural F1. Conclusion We successfully extracted the rF1 with good immunological activity that might be used to detecting Yersinia pestis.