一种用于广泛检测亚洲和非洲寨卡病毒谱系的反转录环介导等温扩增。

A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages.

机构信息

Tropical Infectious Diseases Research and Education Centre (TIDREC), Universiti Malaya, Kuala Lumpur, Malaysia.

Institute for Advanced Studies (IAS), Universiti Malaya, Kuala Lumpur, Malaysia.

出版信息

BMC Infect Dis. 2020 Dec 11;20(1):947. doi: 10.1186/s12879-020-05585-4.

DOI:10.1186/s12879-020-05585-4

PMID:33308203

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7731766/

Abstract

BACKGROUND

Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.

METHODS

In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.

RESULTS

The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6-98.2) and 100% (95% CI = 78.5-100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P < 0.001).

CONCLUSION

The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

摘要

背景

在病毒血症和病毒尿症期间尽早检测寨卡病毒（ZIKV）感染，有助于对患者进行适当的管理，并采取控制蚊虫的措施来防止疾病传播。因此，在资源匮乏的环境中，需要一种经济有效的核酸检测方法来诊断 ZIKV 感染。

方法

本研究开发了一种单管逆转录环介导等温扩增（RT-LAMP）检测法，用于检测亚洲和非洲谱系的 ZIKV。评估了 ZIKV RT-LAMP 检测法的检测限、菌株覆盖率和交叉反应性。使用总共 24 个模拟临床样本评估了 RT-LAMP 的灵敏度和特异性。ZIKV 定量逆转录聚合酶链反应（qRT-PCR）检测法被用作参考检测法。

结果

RT-LAMP 检测法的检测限为 3.73 ZIKV RNA 拷贝（概率分析，P≤0.05）。RT-LAMP 检测法可检测亚洲和非洲谱系的 ZIKV 基因组，而不会与其他节肢动物传播的病毒发生交叉反应。RT-LAMP 检测法的灵敏度和特异性分别为 90%（95%置信区间=59.6-98.2）和 100%（95%置信区间=78.5-100.0）。与 qRT-PCR 检测法相比，RT-LAMP 检测法在 24 个模拟临床样本中的 9 个（37.5%）中检测到 ZIKV 基因组，而在 24 个样本中，qRT-PCR 检测法检测到 10 个（41.7%），具有高度一致性（κ=0.913，P<0.001）。