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- Title
基于毛细管电泳的7种猪源性疫病的多重PCR检测方法的建立.
- Authors
涂藤; 尹清清; 张鹏飞; 王印; 杨泽晓; 姚学萍; 罗燕
- Abstract
The aim of this study was to develop a multiple PCR assay on a basis of capillary electrophoresis for simultaneous detection of seven pathogens of swine infectious diseases, including Streptococcus suis(SS), Classical swine fever virus(CSFV), Japanese encephalitis virus(JEV), Porcine reproductive and respiratory syndrome virus(PRRSV), Haemophilus parasuis(HPS), Porcine rotavirus(PoRV), Salmonella(SE). Firstly the conservative sequences of the 7 reference pathogens were analyzed to design 7 pairs of specific primers. Subsequently, the specific chimeric primers were obtained by adding universal primer sequences in 5' end of each specific primer. Gradient PCR amplification was adopted to optimize annealing temperature. The primer concentrations were optimized by orthogonal test. The sensitivity test was performed by using serially diluted standards (107-10-1 copies·μL-1). Cross reactivity test was performed by using random mixed templates that orginate from cDNA or DNA of these 7 pathogens. Specificity test was performed by detection of some other common pathogens, including PEDV, TGEV, ASFV plasmid, E. coli. Repeatability test was performed by using serially diluted standards as templates to be amplified triplicates. The results indicated that the optimum annealing temperature was 58.3°C. Detection limit of this assay was 10³ copies·μL-1, when 7 positive results were visible from identical lane. With a variety of control samples, the results of positive samples showed specificity peak in contrast of the negative results of all the negative control samples. In addition, neither cross reactivity nor unspecific amplification were detected within these 7 kinds of random mixed templates. the differences among triplicate experiments were small and low. The test results of the 63 clinical samples were exactly the same with the results obtained by national standards. As is indicated in the results, it is an effective toll applied to differential diagnosis rapidly for clinical samples and epidemiological investigation.
- Subjects
CAPILLARY electrophoresis; PATHOGENIC microorganisms; SWINE infections; HAEMOPHILUS parainfluenzae; SALMONELLA
- Publication
Acta Agriculturae Zhejiangensis, 2021, Vol 33, Issue 4, p618
- ISSN
1004-1524
- Publication type
Article
- DOI
10.3969/j.issn.1004-1524.2021.04.07