| Objective: To set up a Multiple-Locus Variable-number tandem-repeat Analysis (MLVA) for molecular typing of Salmonella paratyphoid A (SPA). And to verify the effective value of MLVA for application.Materials and Methods: 21 strains of SPA were bought from Chinese National Center for Medical Culture Collections and 194 clinic strains of SPA which were used in the study of typing analysis were isolated from Yuxi city of Yunnan province from 2005 to 2009. Systemic test was used in the study. Firstly, TRF software was applied to screen and identify the tandem repeat locus according to the sequence of two sequencing strains of SPA, A-9150 and A-12601. Secondly, polymorphism of Variable-number tandem-repeat (VNTR) was screened based on the sequence and sample test. Thirdly, available VNTR were identified with agarose gel electrophoresis, capillary electrophoresis and sequencing. Lastly, the 194 clinic isolates of SPA were typed with the available VNTR. Bionumerics and UPGMA were used for cluster analysis and the analysis results were showed with classification and minimal spanning tree.Results: 1591 and 1512 repeat locus were screened from the genome of A-9150 and A-12601 with TRF software and the polymorphism of repeat number of TR27 was found in those two genome. Then 51 repeat locus were screened according to their TRF score > 80%, the number of copies of repeat locus >3, or sequence homology>85%. Eventually, 9 tandem repeats were identified for typing of SPA by systemic test, they were TR5, TR51, TR24, TR27, TR40, TR41, TR43, TR44, and TR49. There were 14 kinds of MLVA types in 21 reference strains of SPA and 9 kinds of MLVA types in 194 strains of SPA isolated from Yuxi city of Yunnan province. And Hongta district was the endemic area of SPA and type 15 was dominant in all types. The result of minimal spanning tree for analysis of 194 isolates of SPA showed that the representative bacteria were six strains of type 11, two strains of type 16 and two strains of type 14. And three types were found separately, they were strain 09620 which was type 13 from Chengjiang County, strain 08140 which was type 17 from Jiangchuan County and strain 07085 which was type 18 from Hongta district. Compared with reference strains,SPA of Hongta district kept at a distance with strain 50433 from Bulgaria, strain 50508 and strain 50509 from Center for Disease Control and Prevention of Guangdong province. SPA of Xinping County kept at a distance with strain 50002 from Denmark and strain 50504 from Dalian Biology Institute. And all 194 SPA strains weren`t related with strain 50433 from Bulgaria and strain 50002 from Denmark-Shanghai Tongji. There existed three clusters based on strain 08140. The first cluster was type 76 including strain05115, 06036, 09616 09622, 09532 and 09639. They had only one different repeat locus from strain 08140. There were three kinds of status in the second cluster. Strain 50101, 50507, 50078, 50672 and 50434 possessed two different locus of MLVA with strain 08140. Strain 350154 had three different loci with strain 08140. And strain 506745 had five different loci with strain 08140. The third cluster had two different locus with strain 08140 including strain 50501, 50502, 50506, 50001, 9150 (ATCC), 50503 and 50084.Conclusion:1)MLVA was established to type Salmonella paratyphoid A.2)MLVA had a effective value in typing SPA and the dominant strain of SPA and epidemic district of SPA in Yuxi city of Yunnan province were discovered with MLVA.3)The incidence of SPA in Hongta district might play an important role in the incidence of SPA in Yuxi city. So Hongta district was the key area of SPA surveillance.4)The prevention and surveillance of MLVA type 15 of SPA should be emphasized, but that of other types of SPA should not be ignored in Yuxi. |
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