陕西株利什曼原虫ITS-1基因片段序列多态性分析

A study on the detection of Bacillus anthracis with multiplex real-time quantitative PCR using TaqMan-LNA probes

目的分析陕西株利什曼原虫rRNA内转录间隔区间I(ITS-1)序列多态性,探讨利什曼原虫ITS-1分子遗传规律。方法陕西省韩城市犬、白蛉以及人体利什曼原虫阳性标本7份,提取核酸,PCR法扩增ITS-1片段,双向测序、拼接后与文献发表的代表株进行序列比对,构建系统发育树,进行系统发育分析。结果 7份标本均扩增出约320bp的片段,ITS-1序列之间同源性>99%,与国内山丘型疫区虫株高度一致,与荒漠型遗传距离较远。系统发育分析陕西株与婴儿利什曼原虫和杜氏利什曼原虫聚为一类。结论陕西省韩城市分离自病犬、传播媒介白蛉及黑热病患者的利什曼原虫ITS-1序列同源,应为犬源型婴儿利什曼原虫。

Objectives To design a system of multiplex quantitative PCR to detect multiple target genes of Bacillus anthrax,to establish a fast and accurate method of genetic diagnosis,and to solve the problem of cross-reactivity with B.cereus during genetic diagnosis. Methods The pagAgene of the virulent B.anthracis plasmid PX01,the capBgene of the virulent plasmid PX02,and the PL3 gene of the chromosomal prophageλBa03 were selected as target genes to optimize multiplex PCR primers and corresponding TaqMan probe sequences.Probes were labeled with LNA,a fluorescent PCR system was optimized,and a multiplex real-time quantitative PCR system was created using primers for fluorescence quantitative PCR designed using software available online.PCR amplification was performed with 3 pairs of primers to clone the target genes in standard plasmids.B.anthracis,B.cereus,B.thuringiensis,B.mycoides,and common intestinal pathogenic bacteria were serially diluted,and a sensitivity experiment was conducted.Soil and milk powder contaminated with weak B.anthracis were used in a simulated test. Results The 3 target genes（pagA,capB,and PL3）of B.anthracis were quickly detected with multiplex real-time quantitative PCR using TaqMan-LNA probes.Fluorescence quantitative PCR solutions were suitable for reaction with common probes.The cloned standard sequences of pagA,CapB,and PL3 were all correct.The detection limit of the pagAgene was 63 copies/μl,that of the CapBgene was 398 copies/μl,and that of the PL3 gene was 114 copies/μl.The technique only detected B.anthracis genes,and it did not amplify the other 4 Bacillus spp.A simulated test of soil and milk powder yielded positive results.The detection limit of the pagAgene was 6.6×104/ml and that of the PL3 gene was 6.6×10-5/ml. Conclusion TaqMan-LNA multiplex real-time quantitative PCR has wide applicability,it provides rapid detection,and it has a high degree of sensitivity and specificity.This technique can differentiate B.anthracis fromB.cereus,and it can be used to test for a B.anthracisinfection.