用于临床样本中登革病毒血清分型的一步法实时逆转录聚合酶链反应检测
One-step real-time RT-PCR assays for serotyping dengue virus in clinical samples.
作者信息
Alm Erik, Lindegren Gunnel, Falk Kerstin Ingrid, Lagerqvist Nina
机构信息
Department of Microbiology, The Public Health Agency of Sweden, SE-171 82, Solna, Sweden.
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77, Stockholm, Sweden.
出版信息
BMC Infect Dis. 2015 Nov 2;15:493. doi: 10.1186/s12879-015-1226-z.
BACKGROUND
Dengue is one of the leading causes of morbidity in tropical and subtropical regions and infection with any of the four dengue virus serotypes (DENV1-4) result in a wide range of clinical manifestations. Given the geographic expansion of DENV1-4, assays for serotyping are needed to be able to perform surveillance and epidemiological studies. In this study, we describe the design and validation of one-step real-time serotype-specific DENV RT-PCR assays.
METHODS
The DENV1, DENV2, DENV3, and DENV4 RT-PCR assays were designed using all available whole genome DENV sequences in the NCBI nucleotide collection. Because of the high mutation rates of RNA viruses, the assays were performed in singleplex format to enable quick modifications to the primer and probe sequences when new genetic variants emerge. The analytical performance of the RT-PCR assays were evaluated using in vitro transcribed RNA and their specificity was determined by testing 24 DENV isolates, external DENV control panels and RNA preparation of non-DENV flaviviruses and non-dengue clinical samples. Additionally, the clinical performance of the serotype-specific DENV RT-PCR were compared to that of the CDC DENV-1-4 RT-PCR using 85 clinical samples collected from patients presenting with acute dengue.
RESULTS
The RT-PCR assays were found to be specific for their respective serotype and did not cross-react with other flaviviruses or human mRNA. All assays had a linear dynamic range of 10(2) to 10(6) copies/reaction with detection limits between 12 and 44 copies/reaction. When testing sera from 85 confirmed acute dengue cases, the serotype-specific DENV RT-PCR assays had 100 % positive agreement with the FDA-approved CDC DENV-1-4 RT-PCR assay performed in a singleplex format. Additionally 15 samples that tested negative in the CDC DENV-1-4- RT-PCR assay were found positive using the serotype-specific DENV RT-PCR assays.
CONCLUSIONS
Our results suggest that these RT-PCR assays are useful alternatives to existing methods for serotyping DENV in clinical sera.
背景
登革热是热带和亚热带地区发病的主要原因之一，感染四种登革热病毒血清型（DENV1 - 4）中的任何一种都会导致广泛的临床表现。鉴于DENV1 - 4的地理扩张，需要进行血清分型检测以开展监测和流行病学研究。在本研究中，我们描述了一步法实时血清型特异性登革热病毒逆转录聚合酶链反应（RT-PCR）检测方法的设计与验证。
方法
利用美国国立生物技术信息中心（NCBI）核苷酸数据库中所有可用的登革热病毒全基因组序列设计DENV1、DENV2、DENV3和DENV4的RT-PCR检测方法。由于RNA病毒的高突变率，检测以单管形式进行，以便在出现新的基因变异时能够快速修改引物和探针序列。使用体外转录RNA评估RT-PCR检测方法的分析性能，并通过检测24株登革热病毒分离株、外部登革热病毒对照样本以及非登革热黄病毒和非登革热临床样本的RNA制备物来确定其特异性。此外，使用从急性登革热患者收集的85份临床样本，将血清型特异性登革热病毒RT-PCR的临床性能与美国疾病控制与预防中心（CDC）的DENV - 1 - 4 RT-PCR进行比较。
结果
发现RT-PCR检测方法对各自的血清型具有特异性，且不与其他黄病毒或人类mRNA发生交叉反应。所有检测方法的线性动态范围为10（2）至10（6）拷贝/反应，检测限在12至44拷贝/反应之间。在检测85例确诊的急性登革热病例的血清时，血清型特异性登革热病毒RT-PCR检测方法与以单管形式进行的美国食品药品监督管理局（FDA）批准的CDC DENV - 1 - 4 RT-PCR检测方法的阳性一致性为100%。此外，在CDC DENV - 1 - 4 RT-PCR检测中呈阴性反应的15份样本，使用血清型特异性登革热病毒RT-PCR检测方法时呈阳性。
结论
我们的结果表明，这些RT-PCR检测方法是临床血清中登革热病毒血清分型现有方法的有用替代方法。
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