Yenchitsomanus P T, Sricharoen P, Jaruthasana I, Pattanakitsakul S N, Nitayaphan S, Mongkolsapaya J, Malasit P
Medical Molecular Biology Unit, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Southeast Asian J Trop Med Public Health. 1996 Jun;27(2):228-36.
A polymerase chain reaction (PCR) method using sets of newly designed primers for rapid detection and simultaneous identification of dengue virus serotypes was developed and tested. The test is based on two sets of primers specific within the envelope (E) and non-structural (NS1) regions of the dengue-virus genome. Two sets of universal primers that bind to two target sequences which are shared by all the four serotypes of the virus within the E and NS1 regions are used. The resulting products are further amplified by another pair of inner or nested universal primers, which also bind to another set of shared sequences within the E and NS1 regions, respectively. The nested PCR of both the E and NS1 regions can detect dengue virus of all the four serotypes at a sensitivity of 1 plaque forming unit (pfu) or less. For the identification of serotypes, a mixture of four pairs of serotype-specific primers, specific to the E region, was used. The primers have been designed to bind to serotype specific sequences within the regions flanked by the outer universal primers, and giving the amplified products of different sizes, each corresponds to one particular serotype (405 bp for Den1, 346 bp for Den2, 196 bp for Den3, and 143 bp for Den4). A protocol has been developed and successfully applied to detect dengue virus in cell-culture supernatants and patients sera. The technique is simple and rapid, capable of not only detecting the dengue virus but also identifying the serotypes of the virus in clinical specimens.
开发并测试了一种聚合酶链反应（PCR）方法，该方法使用新设计的引物组来快速检测和同时鉴定登革病毒血清型。该测试基于两组针对登革病毒基因组包膜（E）区和非结构（NS1）区的特异性引物。使用两组通用引物，它们分别与病毒所有四种血清型在E区和NS1区内共享的两个靶序列结合。所得产物通过另一对内部或巢式通用引物进一步扩增，这对引物也分别与E区和NS1区内的另一组共享序列结合。E区和NS1区的巢式PCR能够以1个蚀斑形成单位（pfu）或更低的灵敏度检测所有四种血清型的登革病毒。为了鉴定血清型，使用了针对E区的四对血清型特异性引物的混合物。这些引物被设计为与外部通用引物侧翼区域内的血清型特异性序列结合，并产生不同大小的扩增产物，每个产物对应一种特定血清型（登革1型为405bp，登革2型为346bp，登革3型为196bp，登革4型为143bp）。已经开发出一种方案并成功应用于检测细胞培养上清液和患者血清中的登革病毒。该技术简单快速，不仅能够检测登革病毒，还能够鉴定临床标本中病毒的血清型。
