Santos Andrea P, Messick Joanne B, Biondo Alexander W, Oliveira Simone T, Pedralli Viviane, Lasta Camila S, Lacerda Luciana A, Esteves Vanessa S, Hofmann-Lehmann Regina, Willi Barbara, González Félix H D
Departamento de Patologia Clínica, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
Vet Clin Pathol. 2009 Dec;38(4):443-52. doi: 10.1111/j.1939-165X.2009.00158.x. Epub 2009 Jun 22.
'Candidatus Mycoplasma turicensis' (CMtc) is a hemotrophic bacterial species that can, alone or in combination, induce anemia in cats. The diagnostic test of choice for hemoplasma infections is PCR. Conventional PCR assays have been developed for the detection of Mycoplasma haemofelis (Mhf) and 'Candidatus M. haemominutum' (CMhm) but not for CMtc. Although real-time PCR assays have been reported for all of the feline hemoplasmas, the expense of necessary instrumentation precludes its use in Brazil and many other countries.
The goals of this study were to develop and optimize a conventional PCR assay to diagnose CMtc using an internal control to detect false-negative results, and to evaluate the occurrence of CMtc infection in domestic cats from Brazil.
Species-specific primers were designed and a PCR assay was developed for the detection of CMtc 16S rDNA in cat blood. Sensitivity was determined by serial 10-fold dilutions of plasmid and DNA extracted from blood from an experimentally infected cat. EDTA blood samples from 373 cats were collected. DNA was extracted using a silica-based protocol and tested using the PCR assay.
Primer concentration, annealing temperature, and MgCl(2) concentration were optimized in the presence and absence of the internal control. Two samples negative for the internal control were excluded. Of the remaining 371 samples (117 healthy and 254 unhealthy cats), 17 (4.6%) were positive for CMtc.
These results demonstrate the utility of an optimized PCR assay to detect CMtc in feline blood samples. We also report for the first time the prevalence of CMtc infection in domestic cats in Brazil.
“图里西支原体（暂定种）”（CMtc）是一种嗜血性细菌，可单独或联合其他因素导致猫贫血。血支原体感染的首选诊断检测方法是聚合酶链反应（PCR）。已开发出常规PCR检测法用于检测猫血支原体（Mhf）和“溶血微小支原体（暂定种）”（CMhm），但未用于检测CMtc。尽管已报道了针对所有猫血支原体的实时PCR检测法，但所需仪器的费用使其无法在巴西和许多其他国家使用。
本研究的目的是开发并优化一种常规PCR检测法，用于诊断CMtc，并使用内部对照来检测假阴性结果，同时评估巴西家猫中CMtc感染的发生率。
设计了种特异性引物，并开发了一种PCR检测法，用于检测猫血液中的CMtc 16S核糖体DNA。通过对质粒和从实验感染猫的血液中提取的DNA进行连续10倍稀释来确定灵敏度。收集了373只猫的乙二胺四乙酸（EDTA）血样。使用基于硅胶的方法提取DNA，并使用PCR检测法进行检测。
在有和没有内部对照的情况下，对引物浓度、退火温度和氯化镁（MgCl₂）浓度进行了优化。排除了两个内部对照为阴性的样本。在其余371个样本（117只健康猫和254只病猫）中，17个（4.6%）CMtc呈阳性。
这些结果证明了优化后的PCR检测法在检测猫血样中CMtc的实用性。我们还首次报告了巴西家猫中CMtc感染的患病率。
