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首页> 外文期刊>Applied and Environmental Microbiology >Real-Time PCR for Simultaneous Detection and Quantification of Borrelia burgdorferi in Field-Collected Ixodes scapularis Ticks from the Northeastern United States
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Real-Time PCR for Simultaneous Detection and Quantification of Borrelia burgdorferi in Field-Collected Ixodes scapularis Ticks from the Northeastern United States

机译：实时PCR用于同时检测和定量来自美国东北部的田间硬I肩I虫中的疏螺旋体

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The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.
机译：野外采集或实验感染的tick中螺旋体的密度主要通过基于显微镜的测定来估计。在这项研究中，针对 Borrelia burgdorferi 特异的 recA 基因的实时定量PCR（qPCR）协议适用于Lightcycler，用于快速检测和定量莱姆病螺旋体， B 。 burgdorferi ，在田间采集的 sxulars scapularis tick中。 qPCR检测 B 的敏感性。感染tick中的 burgdorferi DNA与针对16S-23S rRNA间隔子的成熟巢式PCR相当。在498个 I 中。从东北四个州（罗德岛州，康涅狄格州，纽约州和新泽西州）收集的 s肩em，438个仙女ny中有91个（60.7％）成年s中有15个（25.0％）成年positive为阳性。 qPCR分析。单个tick中的螺旋体数量从25到197,200不等，每个若虫tick平均为1,964螺旋，每个成年tick平均为5,351螺旋。在这四个州的不同位置（ P = 0.23，通过单向方差分析得出）的locations虫计数或感染tick虫的iro虫计数的平均值均无显着差异。 B 的三种不同的核糖体间隔区限制性片段长度多态性类型。 burgdorferi （ P = 0.39）。观察到感染的tick之间有高度的螺旋体聚集（方均值比为24,877;瞬时估计值 k = 0.279）。从频率分布数据和先前发表的传播研究中，我们估计在宿主宿主的若虫壁虱中可能至少需要300个生物体，才能在以小鼠为食的同时将感染传播给小鼠。这些数据表明，实时定量PCR是同时检测和定量 B 的可靠方法。在田间采集的s中感染 burgdorferi ，可用于莱姆病螺旋体的生态和流行病学监测。

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