Ambient stable quantitative PCR reagents for the detection of Yersinia pestis.
Ambient stable quantitative PCR reagents for the detection of Yersinia pestis.
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DOI：
10.1371/journal.pntd.0000629
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发表时间：
2010-03-09
影响因子：
3.8
通讯作者：
Yang R
中科院分区：
文献类型：
--
作者：
Qu S;Shi Q;Zhou L;Guo Z;Zhou D;Zhai J;Yang R
虽然基于TaqMan探针的实时荧光PCR检测鼠疫耶尔森氏菌的方法已经发展了多年，但室温稳定的PCR试剂的报道很少，这将是非常宝贵的现场疫情监测，公共卫生突发事件的即时反应，反生物恐怖主义调查等。鼠疫杆菌在37°C下具有非凡的稳定性。以Y染色体上的3a序列和质粒pMT 1中的F1抗原基因caf 1为靶点，设计引物和探针，建立了基于TaqMan的实时荧光定量PCR（real-time PCR）方法。鼠疫，分别。然后，将碳水化合物混合物添加到PCR试剂中，随后将其真空干燥以进行稳定性评价。真空干燥的试剂在37°C下对于较低浓度的模板DNA（10拷贝/μl）稳定至少49天，对于较高浓度（≥102拷贝/μl）稳定长达79天。用该试剂检测了土壤样品中的钇。用3a和caf 1引物PCR检测鼠疫疫苗株EV 76，每克土壤可检出5×104 CFU。此外，本文还提出了一种简单有效的土样处理方法。用于实时PCR的真空干燥试剂在37°C下至少保持49天的准确性和再现性，这表明如果可以获得用于进行实时PCR的机器，它们可以在室温下容易地运输用于现场应用。该干试剂对鼠疫常规监测具有重要意义。鼠疫由鼠疫耶尔森氏菌引起，是人类历史上最古老、最危险的疾病之一，在历史上的三次大流行中，夺去了数百万人的生命。虽然黑死病造成的恐慌正在消退，但鼠疫大流行重新出现的威胁仍然存在，还有可能被滥用于生物战或生物恐怖主义。现场快速检测和鉴定病原体对于及时采取有效对策至关重要。基于TaqMan探针的实时PCR检测可以提供快速准确的鉴定;然而，对冷藏和储存的需求阻碍了其潜在的现场应用。本研究的目的是开发一种稳定的PCR系统，便于在室温下运输和储存，这对于常规鼠疫监测和突发公共卫生事件的准备至关重要。我们提出了一个解决方案，希望它有助于未来的应用。
Although assays for detecting Yersinia pestis using TaqMan probe-based real-time PCR have been developed for years, little is reported on room-temperature-stable PCR reagents, which will be invaluable for field epidemic surveillance, immediate response to public health emergencies, counter-bioterrorism investigation, etc. In this work, a set of real-time PCR reagents for rapid detection of Y. pestis was developed with extraordinary stability at 37°C. TaqMan-based real-time PCR assays were developed using the primers and probes targeting the 3a sequence in the chromosome and the F1 antigen gene caf1 in the plasmid pMT1of Y. pestis, respectively. Then, carbohydrate mixtures were added to the PCR reagents, which were later vacuum-dried for stability evaluation. The vacuum-dried reagents were stable at 37°C for at least 49 days for a lower concentration of template DNA (10 copies/µl), and up to 79 days for higher concentrations (≥102 copies/µl). The reagents were used subsequently to detect soil samples spiked with Y. pestis vaccine strain EV76, and 5×104 CFU per gram of soil could be detected by both 3a- and caf1-based PCR reagents. In addition, a simple and efficient method for soil sample processing is presented here. The vacuum-dried reagents for real-time PCR maintain accuracy and reproducibility for at least 49 days at 37°C, indicating that they can be easily transported at room temperature for field application if the machine for performing real-time PCR is available. This dry reagent is of great significance for routine plague surveillance. Plague, caused by Yersinia pestis, is one of the oldest and most dangerous diseases in human history, and has claimed millions of lives in the three major historical pandemics. Although panic caused by the Black Death is fading, the threat of the reemergence of plague pandemics still exists, with the additional potential of misuse in biowarfare or bioterrorism. Rapid on-site detection and identification of the pathogen is of paramount significance for timely implementation of effective countermeasures. TaqMan probe-based real-time PCR assays can give quick and accurate identification; however, the need for cold delivery and storage prevents its potential on-site application. The objective of this study was to develop a stable PCR system for easy delivery and storage under room temperature, which is vital for conventional plague surveillance and for preparedness in public health emergencies. We present a solution to this particular issue, hoping that it is helpful to future applications.
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影响因子：
2.1
作者：
Dohm, DJ;Rowton, ED;Turell, MJ
通讯作者：
Turell, MJ
影响因子：
168.9
作者：
Chanteau, S;Rahalison, L;Nato, A
通讯作者：
Nato, A
DOI：
10.1128/cdli.5.5.609-612.1998
发表时间：
1998-09-01
期刊：
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY
影响因子：
--
作者：
Anderson, GP;King, KD;Ezzell, J
通讯作者：
Ezzell, J
影响因子：
2.6
作者：
Krämer, F;Vollrath, T;Epe, C
通讯作者：
Epe, C
DOI：
10.1111/j.1365-2672.1994.tb01622.x
发表时间：
1994-03-01
期刊：
JOURNAL OF APPLIED BACTERIOLOGY
影响因子：
--
作者：
NORKINA, OV;KULICHENKO, AN;DROSDOV, IG
通讯作者：
DROSDOV, IG