Assessment of a quantitative 5' nuclease real-time polymerase chain reaction using the nicotinamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) for Bartonella species in domiciled and stray cats in Brazil
Assessment of a quantitative 5' nuclease real-time polymerase chain reaction using the nicotinamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) for Bartonella species in domiciled and stray cats in Brazil
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DOI：
10.1177/1098612x15593787
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发表时间：
2016-10-01
影响因子：
1.7
通讯作者：
Machado, Rosangela Zacarias
中科院分区：
文献类型：
--
作者：
Andre, Marcos Rogerio;Dumler, John Stephen;Machado, Rosangela Zacarias
目的建立一种5'核酸酶实时定量聚合酶链反应（5' nuclease real-time polymerase chain reaction，PCR）方法，用于巴尔通体（Bartonella species，简称Bartonella）感染的诊断。对猫血液样品进行针对巴尔通体属物种的靶向烟酰胺腺嘌呤二核苷酸脱氢酶γ亚基（nuoG）的新型定量5'核酸酶实时PCR（qPCR）和靶向基因间转录间隔区、ribC、gltA、pap 31和rpoB的常规PCR测定，结果qPCR检测质粒的拷贝数为10个/反应。151只猫中有46只（54.4%的流浪猫和45.6%的流浪猫）在巴尔通体属的nuoG qPCR中显示阳性结果。采样猫中nuoG巴尔通体DNA的绝对定量范围为1.1 x 10（4）至1.3 x 10（4）。在qPCR检测的46份阳性样本中，有18份（39.1%）在常规PCR检测中也呈阳性。测序证实，汉赛巴尔通体和巴尔通体clarridgeiae循环猫在中西部brazi.Conclusions和相关性-目前的工作提供了一种新的qPCR检测诊断巴尔通体属物种引起的感染的细节。
Objectives The objective of this study was to develop a quantitative 5' nuclease real-time polymerase chain reaction (PCR) assay to diagnose infections caused by Bartonella species.Methods Between January and April 2013 whole blood samples were collected by convenience from 151 cats (86 domiciled and 65 stray cats). The feline blood samples were subjected to a novel quantitative 5' nuclease real-time PCR (qPCR) for Bartonella species targeting the nictonamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) and conventional PCR assays targeting intergenic transcribed spacer, ribC, gltA, pap31 and rpoB, followed by sequencing and basic local alignment search tool analysis.Results The qPCR assay detected as few as 10 copies of plasmid per reaction. Forty-six (54.4% domiciled and 45.6% stray cats) of 151 sampled cats showed positive results in nuoG qPCR for Bartonella species. The absolute quantification of nuoG Bartonella DNA in sampled cats ranged from 1.1 x 10(4) to 1.3 x 10(4). Eighteen (39.1%) of 46 positive samples in the qPCR were also positive in conventional PCR assays. The sequencing confirmed that Bartonella henselae and Bartonella clarridgeiae circulate in cats in midwestern Brazil.Conclusions and relevance The present work provides details of a novel qPCR assay to diagnose infections caused by Bartonella species.