摘要
目的初步了解云南省龙陵县HIV阳性者感染弓形虫的基因型特征。方法使用基因提取试剂盒提取龙陵县HIV阳性者感染弓形虫的基因,运用巢式PCR技术分别对弓形虫SAG2基因(241bp、221bp)进行扩增,扩增产物用1.5%琼脂糖凝胶电泳,紫外投射仪下观察结果。用DNA胶回收试剂盒切胶回收PCR产物并进行酶切,酶切产物置于37℃恒温培养箱孵育16h,孵育后的酶切产物用2.5%琼脂糖凝胶电泳,凝胶成像系统摄像观察结果并与弓形虫基因Ⅰ型标准株进行对比鉴定。结果 112份HIV阳性者全血37份扩增出弓形虫SAG2基因(241bp、221bp)。从37份弓形虫SAG2基因(241bp、221bp)扩增阳性的全血标本中选择条带较亮的6份进行酶切,并与弓形虫基因I型标准株比对,初步确定2号标本为基因Ⅲ型,其他标本为基因Ⅰ型。从酶切的6份标本中选择2份进行测序,其中1份为酶切Ⅲ型(测序的2号标本)。经过基因序列对比分析,各弓形虫株之间SAG2基因的碱基对差异较小。其中基因Ⅰ型36份,Ⅲ型1份。结论初步确定云南省龙陵县HIV阳性者感染弓形虫的基因型以Ⅰ型为主,Ⅲ型少见,未见Ⅱ型及其他基因型。
Objective To obtain a preliminary understanding of the genotype characteristics of Toxoplasma gondii infecting people with HIV in Longling County, Yunnan Province. Methods T. gondii genes were extracted with a DNA extraction kit from people with HIV in Longling County. Nested PCR was used to amplify the SAG2 genes of T. gondii （241 bp and 221 bp）. Amplification products were subjected to 1.5% agarose gel electrophoresis and observed under a UV projector. A DNA Gel Extraction Kit was used to digest recovered PCR products, and digestion products were incubated in an incubator at 37 C for 16 h. After incubation, the digestion products were subjected to 2.5% agarose gel electrophoresis. T. gondii was identified in comparison to a standard strain （genotype I） and imaging results from a gel imaging system. Results SAG2 genes of T. gondii （241 bp and 221 bp） were amplified from 37 blood samples from 112 individuals with HIV. Of the 37 blood samples with the SAG2 genes of T. gondii （241 bp and 221 bp）, 6 samples produced distinct bands. These samples were digested and compared to the standard strain. Strains in sample 2 were preliminarily identified as genotype Ⅲ while those in the other samples were genotype I. Two of the 6 samples that were digested were sequenced, and 1 of the 2 samples was identified as genotype Ⅲ （sample 2 in sequencing） upon digestion. Gene sequencing indicated that base pairs differed little in the strains of the SAG2 gene of T. gondii. Thirty-six of the samples were genotype I and 1 sample was genotype Ⅲ. Conclusion The genotype of T. gondii infecting HIV-positive individuals in Longling County, Yunnan Province was preliminarily determined to be mostly genotype Ⅰ. Genotype Ⅲ was also present but rare, and genotype Ⅱand other genotypes were not detected.
出处
《中国病原生物学杂志》
CSCD
北大核心
2014年第5期438-441,446,共5页
Journal of Pathogen Biology
关键词
HIV阳性
弓形虫
SAG2基因
PCR^RFLP
序列分析
HIV-positive, Toxoplasma gondii, SAG2 gene, PCR-linked restriction fragment length polymorphism,
