Rodríguez-Lázaro David, Lewis Deborah A, Ocampo-Sosa Alain A, Fogarty Ursula, Makrai László, Navas Jesús, Scortti Mariela, Hernández Marta, Vázquez-Boland José A
Bacterial Molecular Pathogenesis Group, Faculty of Medical and Veterinary Sciences, University of Bristol, Langford, United Kingdom.
Appl Environ Microbiol. 2006 Jun;72(6):4256-63. doi: 10.1128/AEM.02706-05.
We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA+ and vapA-negative (including vapB+) plasmid genotypes. The vapA+ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to approximately 10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA+) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA+ "horse-pathogenic" genotype determination.
我们开发了一种针对土壤放线菌马红球菌的新型定量实时PCR(Q-PCR)方法,马红球菌是一种重要的马匹病原体和新出现的人类病原体。通过靶向马红球菌中普遍保守的染色体单拷贝基因choE实现了物种特异性定量。choE Q-PCR包含一个内部扩增对照(IAC),用于识别假阴性。第二个Q-PCR靶向毒力质粒基因vapA,大多数马匹分离株携带该基因,但在其他来源的分离株中很少发现。使用178株马红球菌分离株、77株非靶细菌以及一组60株已知vapA+和vapA阴性(包括vapB+)质粒基因型的马红球菌分离株进行测定,结果表明choE-IAC和vapA检测方法的敏感性和特异性均为100%。不同类型分离株中vapA+的频率如下:马匹,85%;人类,20%;牛和猪,0%;其他,27%。choE-IAC Q-PCR使用马红球菌DNA时可检测到低至一个基因组当量,使用从人工污染的马支气管肺泡灌洗(BAL)液中提取的DNA时可检测到100个细菌/ml。在6个对数的动态范围内,定量呈线性,低至约10个靶分子(或1000 CFU/ml BAL液),PCR效率E>0.94。vapA检测具有相似的性能,但由于靶基因或质粒拷贝数(1至9)的变异性,似乎不适用于准确的(vapA+)马红球菌定量。本文报道的双反应Q-PCR系统为医学和兽医诊断实验室提供了一种有用的工具,用于马红球菌的定量检测和(可选)vapA+“马致病性”基因型的测定。