一种用于登革热感染早期诊断的基于新型逆转录酶-聚合酶链反应的液体杂交（RT-PCR-LH）检测方法。

A novel reverse transcriptase-polymerase chain reaction based-liquid hybridisation (RT-PCR-LH) assay for early diagnosis of dengue infection.

作者信息

Gunesekera Maya B, Hapugoda Menaka D, Gunasena S, Subasinghe S A S C, Bandara K B A T, Khan Baldip K, Abeyewickreme W

机构信息

Department of Chemistry, Faculty of Science, University of Colombo, Colombo 3.

出版信息

Ceylon Med J. 2003 Mar;48(1):17-22. doi: 10.4038/cmj.v48i1.3389.

DOI:10.4038/cmj.v48i1.3389

PMID:12795015

Abstract

BACKGROUND

Early definitive laboratory diagnosis of dengue is difficult with the tests in routine use at present.

OBJECTIVE

To develop a reverse transcriptase-polymerase chain reaction based liquid hybridisation (RT-PCR-LH) technique for the rapid and early diagnosis of dengue.

RESEARCH DESIGN

RT-PCR products of the NS3 gene of dengue virus prototypes and of a few positive sera for dengue virus by culture, were allowed to hybridise in liquid phase with a mixture of dengue specific radiolabelled oligonucleotides. The products were separated by PAGE and visualised by autoradiography. 78 suspected dengue sera were also tested by RT-PCR-LH method, and by IgM-ELISA and HAI tests, for comparison.

RESULTS

Two DNA bands (approximately equal to 470 bp and approximately equal to 455 bp) specific to dengue virus, were observed. RT-PCR-LH assay takes only 24 h. Of the 78 suspected dengue acute sera tested, 45/78 were positive by RT-PCR-LH, 31/78 were positive by IgM-ELISA, and 14/78 had a HAI titre > or = 2560. Duration of fever was known in 72 cases, and infection was detected by RT-PCR-LH in 11/22 of cases with < 5 d fever and by IgM-ELISA in 1/22. In cases with 5 to 15 d fever RT-PCR-LH and IgM-ELISA/HAI titre > or = 2560 detected infection in 30/50 and 27/50 respectively. The 10 sera which were negative by RT-PCR-LH, but were positive by either IgM-ELISA or HAI titre > or = 2560 were all > 5 d fever cases. RT-PCR-LH together with IgM-ELISA were capable of detecting dengue infection in 56/78 of the suspected cases.

CONCLUSION

RT-PCR-LH assay developed in this study appears to have an advantage over other diagnostic techniques for the early detection of dengue.

摘要

背景

目前常规使用的检测方法难以对登革热进行早期明确的实验室诊断。

目的

开发一种基于逆转录酶-聚合酶链反应的液相杂交（RT-PCR-LH）技术，用于登革热的快速早期诊断。

研究设计

登革热病毒原型的NS3基因的RT-PCR产物以及通过培养获得的一些登革热病毒阳性血清，使其与登革热特异性放射性标记寡核苷酸混合物在液相中杂交。产物通过聚丙烯酰胺凝胶电泳（PAGE）分离并通过放射自显影进行可视化。还采用RT-PCR-LH方法以及IgM-ELISA和血凝抑制试验（HAI）对78份疑似登革热血清进行检测，以作比较。

结果

观察到两条登革热病毒特异性DNA条带（约470 bp和约455 bp）。RT-PCR-LH检测仅需24小时。在检测的78份疑似登革热急性血清中，RT-PCR-LH检测阳性的有45/78，IgM-ELISA检测阳性的有31/78，血凝抑制试验效价≥2560的有14/78。已知72例患者的发热持续时间，在发热＜5天的病例中，RT-PCR-LH检测出11/22例感染，IgM-ELISA检测出1/22例。在发热5至15天的病例中，RT-PCR-LH和IgM-ELISA/血凝抑制试验效价≥2560分别检测出30/50例和27/50例感染。RT-PCR-LH检测为阴性但IgM-ELISA或血凝抑制试验效价≥2560为阳性的10份血清均为发热＞5天的病例。RT-PCR-LH与IgM-ELISA联合能够在56/78例疑似病例中检测出登革热感染。