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首页> 外文期刊>Journal of Clinical Microbiology >Development of type-specific PCR for typing Pneumocystis carinii f. sp. hominis based on nucleotide sequence variations of internal transcribed spacer region of rRNA genes.
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Development of type-specific PCR for typing Pneumocystis carinii f. sp. hominis based on nucleotide sequence variations of internal transcribed spacer region of rRNA genes.

机译：开发用于分型卡氏肺孢子虫的类型特异性PCR。 sp。基于rRNA基因内部转录间隔区核苷酸序列的变异

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The nucleotide sequence variations in the internal transcribed spacer region 1 (ITS1) and region 2 (ITS2) of rRNA genes were found to be useful for typing Pneumocystis carinii isolates that infect humans. Two types of ITS1 (A and B) and three types of ITS2 (a, b, and c) sequences have been found, and P. carinii isolates are classified based on sequence types of ITS1 and ITS2 as Ax or Bx (where x may be a, b, or c). Type determination has been achieved by sequencing the ITS regions or by reacting the ITS regions amplified by PCR with type-specific oligonucleotide (TSO) probes. However, TSO typing alone does not work on a specimen from an individual who is infected by more than one strain of P. carinii where different ITS1 types are present in the same specimen. In this study, type-specific PCR assays were developed to supplement TSO typing. Type-specific PCR primers were made so that they differ at their 3' ends by the two nucleotides which distinguish type A from type B of ITS1 plus an additional "A" residue at the extreme 3' ends of the primers. These two primers were paired separately with a general primer which anneals to a region downstream from ITS2 to specifically amplify Ax or Bx. The amplified products were then reacted separately with ITS2-specific probes 2-a, 2-b, and 2-c to identify their types.
机译：发现rRNA基因的内部转录间隔区1（ITS1）和区域2（ITS2）中的核苷酸序列变异可用于分型感染人类的​​卡氏肺孢子虫分离株。发现了两种类型的ITS1（A和B）和三种类型的ITS2（a，b和c）序列，并且根据ITS1和ITS2的序列类型将卡氏疟原虫分离株分类为Ax或Bx（其中x是a，b或c）。通过对ITS区测序或通过PCR扩增的ITS区与类型特异性寡核苷酸（TSO）探针反应，可以实现类型确定。但是，仅TSO分型不适用于感染了一种以上卡氏疟原虫菌株的个体的标本，而同一样本中存在不同的ITS1类型。在这项研究中，开发了类型特异性PCR分析法以补充TSO分型。制备类型特异性PCR引物，以使它们在3'末端的区别在于两个核苷酸，这两个核苷酸区分ITS1的A型与B型，并在引物的3'末端带有一个额外的“ A”残基。这两个引物分别与退火至ITS2下游区域的通用引物配对，以特异性扩增Ax或Bx。然后将扩增产物分别与ITS2特异性探针2-a，2-b和2-c反应以鉴定其类型。

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