Maneekarn N, Morita K, Tanaka M, Igarashi A, Usawattanakul W, Sirisanthana V, Innis B L, Sittisombut N, Nisalak A, Nimmanitya S
Department of Microbiology, Faculty of Medicine, Chiang Mai University, Thailand.
Microbiol Immunol. 1993;37(1):41-7. doi: 10.1111/j.1348-0421.1993.tb03177.x.
A simple and sensitive procedure of reverse transcriptase polymerase chain reaction (RT-PCR) was developed previously such that all 4 serotypes of dengue viruses could be detected and their serotypes identified simultaneously in a single-step procedure. In this study we compared the RT-PCR with a conventional immunoperoxidase (PAP) staining method for the identification of dengue viruses currently isolated from patient sera. Sixty-six sera taken from dengue hemorrhagic fever (DHF) patients were subjected to virus isolation by inoculating onto C6/36 cell cultures. Screening for the presence of dengue viruses in culture fluids was done after 7 days of incubation by PAP staining using hyperimmune rabbit anti-dengue virus antibody as the primary reagent. Dengue viruses in positive cultures were further identified for their serotypes by PAP using type-specific monoclonal antibodies (MAb) and by RT-PCR. Thirty-two out of the 66 serum specimens tested (48.5%) were positive for dengue viruses. Of these, 5 were type 1 (DEN-1), 25 were type 2 (DEN-2) and 2 contained both DEN-1 and DEN-2. All cultures that were positive by PAP method were also positive by RT-PCR and vice versa. Thus, the results obtained by RT-PCR were in good agreement with those by PAP. It is important to point out that while all 5 DEN-1 isolates reacted readily with the MAb 1F1, only 2 of them could be identified by the MAb 15F3. Our data suggest that antigenic variation among DEN-1 isolates occur frequently and this should be taken into consideration in the selection of appropriate type-specific MAb for serotyping of dengue viruses.(ABSTRACT TRUNCATED AT 250 WORDS)
先前已开发出一种简单且灵敏的逆转录聚合酶链反应（RT-PCR）方法，可在一步操作中同时检测登革病毒的所有4种血清型并鉴定其血清型。在本研究中，我们将RT-PCR与传统免疫过氧化物酶（PAP）染色方法进行比较，以鉴定目前从患者血清中分离出的登革病毒。从登革出血热（DHF）患者采集的66份血清接种到C6/36细胞培养物中进行病毒分离。培养7天后，使用超免疫兔抗登革病毒抗体作为主要试剂，通过PAP染色筛查培养液中是否存在登革病毒。阳性培养物中的登革病毒通过使用型特异性单克隆抗体（MAb）的PAP和RT-PCR进一步鉴定血清型。66份检测的血清标本中有32份（48.5%）登革病毒呈阳性。其中，5份为1型（DEN-1），25份为2型（DEN-2），2份同时含有DEN-1和DEN-2。所有通过PAP方法呈阳性的培养物通过RT-PCR也呈阳性，反之亦然。因此，RT-PCR获得的结果与PAP的结果高度一致。需要指出的是，虽然所有5株DEN-1分离株都能与单克隆抗体1F1迅速反应，但其中只有2株能被单克隆抗体15F3鉴定。我们的数据表明，DEN-1分离株之间频繁发生抗原变异，在选择合适的型特异性单克隆抗体进行登革病毒血清分型时应考虑这一点。（摘要截短至250字）
