Rapid Real-Time PCR Assays for Detection of Klebsiella pneumoniae with the rmpA or magA Genes Associated with the Hypermucoviscosity Phenotype Screening of Nonhuman Primates
Rapid Real-Time PCR Assays for Detection of Klebsiella pneumoniae with the rmpA or magA Genes Associated with the Hypermucoviscosity Phenotype Screening of Nonhuman Primates
复制标题
DOI：
10.2353/jmoldx.2009.080136
复制
发表时间：
2009-09-01
影响因子：
4.1
通讯作者：
Kulesh, David A.
中科院分区：
文献类型：
--
作者：
Hartman, Laurie J.;Selby, Edward B.;Kulesh, David A.
黏液黏度相关基因（magA）和/或黏液样表型调节基因（rmpA）与肺炎克雷伯菌高黏液黏度（HMV）表型的关系已被报道。我们先前证明rmpA +肺炎克雷伯菌可在非洲绿猴中引起严重疾病，并从其他非人灵长类动物中分离出rmpA +和magA + HMV肺炎克雷伯菌。为了快速筛选非洲绿猴/非人类灵长类动物的这些感染，我们开发了三种实时PCR检测方法。第一种是针对肺炎克雷伯菌的，靶向khe基因，而其他两种靶向rmpA和magA基因。利用引物表达2与三个肺炎克雷伯菌基因建立序列特异性的Taq-Man/TaqMan-Minor凹槽结合物检测。采集口腔/直肠拭子和尸检样本；拭子用于常规培养和DNA提取。使用肺炎克雷伯菌特异性测定法对Vitek 2进行DNA检测，鉴定出肺炎克雷伯菌菌落。对45只非洲绿猴的测试结果显示，来自14只动物的19个khe+样本均未对rmpA或magA呈阳性反应。在这19个khe +样本中，5个培养阳性，但没有一个HMV“串试验”阳性。随后对307只非人灵长类动物的检测结果为64株HMV肺炎克雷伯菌分离株，其中42株为rmpA +， 15株为magA +。在美国陆军传染病医学研究所进行的非人类灵长类动物试验表明，通过培养和实时聚合酶链反应（real-time PCR）来确定HMV基因型，能够筛选活的和死的动物是否存在肺炎克雷伯菌。（分子诊断杂志，2009,11:464-471;DOI: 10.2353/jmoldx.2009.080136）
The relationship of mucoviscosity-associated (magA) and/or regulator of mucoid phenotype (rmpA) genes to the Klebsiella pneumoniae hypermucoviscosity (HMV) phenotype has been reported. We previously demonstrated that rmpA + K. pneumoniae can cause serious disease in African green monkeys and isolated rmpA + and magA + HMV K. pneumoniae from other species of non-human primates. To rapidly screen African green monkeys/non-human primates for these infections, we developed three real-time PCR assays. The first was K. pneumoniae-specific, targeting the khe gene, while the others targeted rmpA and magA. Primer Express 2 was used with the three K. pneumoniae genes to generate sequence-specific Taq-Man/TaqMan-Minor Groove Binder assays. Oral/rectal swabs and necropsy samples were collected; swabs were used for routine culture and DNA extraction. K. pneumoniae colonies were identified on the Vitek 2 with DNA tested using the K. pneumoniae-specific assays. Testing of 45 African green monkeys resulted in 19 khe+ samples from 14 animals with none positive for either rmpA or magA. Of these 19 khe + samples, five were culture-positive, but none were HMV "string test"-positive. Subsequent testing of 307 non-human primates resulted in 64 HMV K. pneumoniae isolates of which 42 were rmpA + and 15 were magA +. Non-human primate testing at the U.S. Army Medical Research institute of Infectious Diseases demonstrated the ability to screen both live and necropsied animals for K. pneumoniae by culture and real-time PCR to determine HMV genotype. (J Mol Diagn 2009,11:464-471; DOI: 10.2353/jmoldx.2009.080136)