Sheikh Nazish, Kumar Sanjay, Sharma Harsh Kumar, Bhagyawant Sameer S, Thavaselvam Duraipandian
Biodetector Development Test and Evaluation Division, Defence Research and Development Establishment, Gwalior, India.
Division of Veterinary Public Health and Epidemiology, Sher-e-Kashmir University of Agricultural Sciences and Technology (SKUAST), Jammu, India.
Front Cell Infect Microbiol. 2020 May 5;10:127. doi: 10.3389/fcimb.2020.00127. eCollection 2020.
Q fever is an important zoonotic disease caused by the bacterium . The agent is considered as a potential agent for bioterrorism because of its low infectious dose, aerial route of transmission, resistance to drying, and many commonly used disinfectants. Humans are largely infected by the inhalation of aerosols that are contaminated with parturition products of infected animals as well as by the consumption of unpasteurized milk products. Thus, rapid and accurate detection of in shedders, especially those that are asymptomatic, is important for early warning, which allows controlling its spread among animals and animal-to-human transmission. In the present study, a colorimetric loop-mediated isothermal amplification (LAMP) assay was developed to confirm the presence of 1111a gene of in sheep vaginal swabs. The sensitivity of this assay was found to be very comparable to the quantitative PCR (qPCR) assay, which could detect three copies of the gene, which corresponds to a single cell of . The applicability of the colorimetric LAMP assay in the disease diagnosis was assessed by evaluating 145 vaginal swab samples collected from the sheep breeding farms with a history of stillbirth and repeated abortions. Compared to qPCR, colorimetric LAMP had a sensitivity of 93.75% (CI, 69.77-99.84%) and specificity of 100% (CI, 97.20-100%), with a positive (PPV) and negative predictive value (NPV) of 100 and 99.24%, respectively. A very high level of agreement was observed between both colorimetric LAMP and reference qPCR assay. The colorimetric LAMP assay reported here is a rapid and simple test without extensive sample preparation and has a short turnaround time of <45 min. To the best of our understanding, it is the very first study describing the use of colorimetric LAMP assay that detects in vaginal swab samples with minimal sample processing for DNA extraction.
Q热是一种由细菌引起的重要人畜共患病。由于其感染剂量低、空气传播途径、耐干燥以及对许多常用消毒剂具有抗性，该病原体被视为生物恐怖主义的潜在病原体。人类主要通过吸入被感染动物分娩产物污染的气溶胶以及食用未巴氏消毒的奶制品而感染。因此，快速准确地检测出 shedding者，尤其是无症状者，对于早期预警非常重要，这有助于控制其在动物间的传播以及动物与人之间的传播。在本研究中，开发了一种比色环介导等温扩增（LAMP）检测方法，以确认绵羊阴道拭子中 1111a基因的存在。发现该检测方法的灵敏度与定量PCR（qPCR）检测方法非常相当，后者可以检测到该基因的三个拷贝，这相当于一个 的单细胞。通过评估从有死胎和反复流产病史的绵羊养殖场采集的145份阴道拭子样本，评估了比色LAMP检测方法在疾病诊断中的适用性。与qPCR相比，比色LAMP的灵敏度为93.75%（CI，69.77 - 99.84%），特异性为100%（CI，97.20 - 100%），阳性预测值（PPV）和阴性预测值（NPV）分别为100%和99.24%。比色LAMP和参考qPCR检测方法之间观察到非常高的一致性。本文报道的比色LAMP检测方法是一种快速简单的检测方法，无需大量样本制备，周转时间短，<45分钟。据我们所知，这是第一项描述使用比色LAMP检测方法在阴道拭子样本中以最少的样本处理进行DNA提取来检测 的研究。
