| Yersinia enterocolitica, Clostridium perfringens and Listeria monocytogenes are three common pathogenic bacteria in foodborne diseases, and food poisoning on people or animals is prevalent in China. Pestis plague caused by Yersinia pestis is a serious, infectious disease. In recent years, different kinds of diseases caused by these 4 pathogens are more and more severe, which have become a serious threat to human health and the stockbreeding. Considering the above, many countries began researches on the method for detection of these pathogens. Nowadays, the xMAP liquid chip techonology has been used in many fields, but it is just started to be researched in China, and hasn't obtained enough consideration. To develop a new rapid, sensitive, multiplexed technology for the detection and identification of pathogens, we carried out the research on detection and identification of 4 pathogens by the xMAP liquid chip technology. This could provide not only a new method for lab research and clinical detection, but also an effective preventation and control of infectious diseases.By searching target gene sequences in GenBank, four sets of long oligonucleotide primers were chosen from ail gene of Yersinia enterocolitica, hly gene of Listeria monocytogenes, cpe gene of Clostridium perfringens, and 3a gene of Yersinia Pestis respectively. The 4 gene long fragments were synthesized by overlapping PCR, and we got 555 bp, 591 bp, 450 bp and 330 bp fragments, which were inserted into pGEM-T or pMD18-T vectors, then transformed into dominant bacteria E. Coli DH5α.We judged the correct cloning vectors by the identification of PCR, the identification of recombinant plasmid with restriction digestion and the analysis of sequences. Respectively, 1 pair of specific primers and 1 probe were chosen from the target fragments we got before. Using the constructed plasmid DNA as the template, the 4 short gene fragments were simultaneously amplified by the multiplex PCR, and the PCR conditions were optimized. We detected the multiplex PCR product from the plasmid DNA by the xMAP liquid chip technology, in which, the reaction condition was analyzed and optimized. Then we detected the multiplex PCR products from the pathogens genomic DNA, and identified the specificity and sensitivity of this method.Four recombinant plasmids and the multiplex PCR production method of 4 pathogens templates were successfully constructed. The multiplex PCR products amplified from the plasmid DNA and the bacterial genomic DNA were detected by the xMAP liquid chip technology, and the results were similar to the multiplex PCR. This new technology, which was more specific and sensitive than PCR and multiplex PCR could be valuable for simultaneous detection of four pathogens by one reaction in a sigle vessel within 3.5 h. In the single and multiplex detection of pathogen genomic DNA, the sensitivity was up to 30 CFU/ml for Yersinia pestis and 20 CFU/ml for Listeria monocytogenes. Finally, we got 4 kinds of diagnostic microspheres for 4 pathogens in our work. In addition, the xMAP liquid chip technology based on the specific nucleic probes was successfully developed for the rapid, multiplex detection and identification of pathogens.The xMAP technology developed in this study could be valuable for exact and rapid detection of the mentioned 4 pathogens. The results indicate that the xMAP liquid chip technology is capable of simultaneously detecting many different analytes in a single reaction vessel. What is more, it possesses the feature of multi-analytes, high sensitivity, good reproducibility, wide channel, simple as well as economical. Compared with the traditional detective method, this new one bares obvious benefits, so it's easily accepted and applied in clinical diagnosis. The xMAP liquid chip technology provides a more efficient and useful method for the rapid, multiplex detection of pathogens, and it will be an important detective method in the research field of biology. Further more, it indicates that the xMAP liquid chip method is valuable for exploitation and application. |
