使用RT-Seminested PCR方法直接由檢體偵測並鑑定 北台灣腸病毒

Direct Detection and Identification of Enterovirus Collected from Clinical Specimens in Northern Taiwan by RT-Seminested PCR

鄭惠玲(Hui-Ling Cheng)

指導教授 ： 高全良

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.00420

摘要

腸病毒(Human enterovirus, HEV)是屬於微小RNA病毒科(picornaviridae family)的病毒，可以分為四群，分別是HEV-A, B, C, D，現在已知至少有八十七種血清型(serotype)被鑑定出來。目前臨床實驗室偵測及鑑定腸病毒血清型主要是以病毒培養分離出的病毒再進一步鑑定型別，由台灣 2000~2008 年六月底前的腸病毒統計來看，以病毒培養方式偵測並鑑定出的腸病毒，只有24種血清型被報導過，除了因為有些腸病毒仍無法在細胞中複製之外，是否因為受限於低病毒離率之故，使得無法完整了解台灣腸病毒流行的真實情況。因分子檢測法比病毒培養檢出率高，所以直接由檢體檢測腸病毒核酸的存在，是快速方便的方法。
本研究利用2006 年Nix 團隊發展的檢測法- VP1 RT-snPCR (CODEHOP)，進行台灣北部地區病毒的檢測及鑑定，(1)盼藉由檢測病毒培養結果為陰性的檢體，了解是否有未被分離出之病毒存在；(2)並探討其血清型與疾病的相關性；(3)尋找是否有未曾發表過之其他血清型；(4)尋找國內外皆未曾發表過之其他血清型。實驗總共檢測了177 件病毒培養為陰性的檢體，VP1 RT-snPCR (CODEHOP)檢測法的陽性檢出率(67.2%) 比5’NTR-nPCR (46.9%)檢測法高，且也比一般病毒培養敏感，證明VP1 RT-snPCR (CODEHOP)確實比較敏感也可以增加腸病毒的檢出率。119 件陽性檢體中，55 件可以鑑定型別，主要為CVA24 variant, CVA6, CVA16,及CVA10。其中一件檢體分析其VP1 的全長基因，與標準株的核苷酸相似度，發現其應是新的血清型腸病毒候選株。另外有64 件定序訊號無法判讀的檢體，則使用CODEHOP HEV-A, B, C screening 檢測法進行篩檢，有39 件為陽性，可以直接用於分型，其中混合感染(mix infection) 就佔了10 件。雖然CODEHOP HEV-A, B, C敏感度比VP1 RT-snPCR(CODEHOP)差，但對於混合感染的檢體可以有效分群並可以進一步定序鑑定出型別。
陰性檢體分離出來最多的血清型為CVA24 variant，主要會引起急性出血性結膜炎，而在這之前，未曾有研究指出CVA24 variant 會造成神經方面相關的疾病，但是由本研究結果發現，在懷疑是腦炎及無菌性腦膜炎病人的CSF 中檢體可以偵測到CVA24 variant，這是否表示CVA24 variant 除了引起急性出血性結膜炎之外，亦會造成其他的疾病，是值得持續觀察的。

關鍵字

陰性檢體 ； 新型腸病毒 ； 混合感染

並列摘要

Human enterovirus (HEV) is a genus of the Picornaviridae including more than 87 serotypes belonging to four species designed Human enterovirus A to D. The diagnosis of enterovirus infections, and in particular the characterization of enterovirus strains, relies on the isolation of virus in cell culture from clinical samples. Based cell culture method, there were 24 serotypes of human enteroviruses reported in Taiwan, 2000-June, 2008. Because many enteroviruses do not grow readily in cell culture and that was limited to the low sensitivity, make us unable to study enterovirus prevalence in Taiwan completely. Hence, diagnostic tests based on PCR are developed and found that it is more sensitive and much faster than conventional culture method.
This study has planned to use the method-VP1 RT-snPCR (CODEHOP) that developed by Nix and his coworkers in 2006 to detect and identify enteroviruses in northern Taiwan. The specific aims of study are: (1) To detect and identify HEV that could not be isolated by virus culture; (2) To analysis the association between disease and HEV infection; (3) To identify possible new serotypes that haven’t been reported in Taiwan; (4) To identify possible new serotypes that haven’t been reported internationally.
177 virus-culture negative specimens were collected and tested by VP1 RT-snPCR (CODEHOP) and 5’ NTR RT-nPCR methods. VP1 RT-snPCR (CODEHOP) method (67.2%) was more sensitive than 5’ NTR RT-nPCR method (47.9%). Also, VP1
RT-snPCR (CODEHOP) method was more sensitive than virus culture and could increase the positive rate of EV detection. Among 119 CODEHOP-positive samples, 55
EV were identified as CVA24 variant, CVA6, CVA16, and CVA10 mainly by partial VP1 sequence analysis.
A new EV serotype candidate was found. The identity of complete VP1 gene of this candidate was less than 70% when compared with reference strains. In addition, there
were still 64 EV-positive results that sequence signal were unable to interpret. Therefore, a new method, called CODEHOP HEV-A, B, C screening method was designed to
solve those problems. The result show that 39 of 64 specimens were detected by this new method and all positive results could be directly grouped. Among 39 positive samples, there were 10 positive results show mix infection. Moreover the sequence signals were all good to interpret. Although CODEHOP HEV-A, B, C screening method
was less sensitive than VP1 RT-snPCR (CODEHOP), it could identify the specimens with two or three serotypes of enteroviruses mix infection.
Serotype, CVA24 variant, usually causes acute hemorrhagic conjunctivitis and was never reported association with neural diseases. In this study, there were many cases identified as CVA24 variant through the detection of virus-culture negative specimens.
Particularly, CVA24 variants were isolated from the CSF specimens of patients diagnosed with encephalitis and aseptic meningitis. It suggested that CVA24 variant
was concerned as the pathogen of neural diseases.

並列關鍵字

CODEHOP HEV-ABC screening method ； mix infection

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