Ahamed Syed Fazil, Vivek Rosario, Kotabagi Shalini, Nayak Kaustuv, Chandele Anmol, Kaja Murali-Krishna, Shet Anita
Division of Infectious Diseases, St. John's Research Institute, St. John's National Academy of Health Sciences, Koramangala, Bangalore, 560034, India.
ICGEB-Emory Vaccine Centre, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
J Virol Methods. 2017 Jun;244:46-54. doi: 10.1016/j.jviromet.2017.02.014. Epub 2017 Feb 28.
Dengue surveillance relies on reverse transcription-polymerase chain reaction (RT-PCR), for confirmation of dengue virus (DENV) serotypes. We compared efficacies of published and modified primer sets targeting envelope (Env) and capsid-premembrane (C-prM) genes for detection of circulating DENV serotypes in southern India. Acute samples from children with clinically-diagnosed dengue were used for RT-PCR testing. All samples were also subjected to dengue serology (NS1 antigen and anti-dengue-IgM/IgG rapid immunochromatographic assay). Nested RT-PCR was performed on viral RNA using three methods targeting 654bp C-prM, 511bp C-prM and 641bp Env regions, respectively. RT-PCR-positive samples were validated by population sequencing. Among 171 children with suspected dengue, 121 were dengue serology-positive and 50 were dengue serology-negative. Among 121 serology-positives, RT-PCR detected 91 (75.2%) by CprM654, 72 (59.5%) by CprM511, and 74 (61.1%) by Env641. Among 50 serology-negatives, 10 (20.0%) were detected by CprM654, 12 (24.0%) by CprM511, and 11 (22.0%) by Env641. Overall detection rate using three methods sequentially was 82.6% (100/121) among serology-positive and 40.0% (20/50) among serology-negative samples; 6.6% (8/120) had co-infection with multiple DENV serotypes. We conclude that detection of acute dengue was enhanced by a modified RT-PCR method targeting the 654bp C-prM region, and further improved by using all three methods sequentially.
登革热监测依赖于逆转录-聚合酶链反应（RT-PCR）来确认登革热病毒（DENV）血清型。我们比较了已发表的和改良的针对包膜（Env）基因和衣壳-前膜（C-prM）基因的引物组在检测印度南部流行的DENV血清型方面的效能。来自临床诊断为登革热的儿童的急性样本用于RT-PCR检测。所有样本还进行了登革热血清学检测（NS1抗原和抗登革热-IgM/IgG快速免疫层析法）。使用分别针对654bp C-prM、511bp C-prM和641bp Env区域的三种方法对病毒RNA进行巢式RT-PCR。RT-PCR阳性样本通过群体测序进行验证。在171名疑似登革热的儿童中，121名登革热血清学检测呈阳性，50名登革热血清学检测呈阴性。在121名血清学阳性者中，RT-PCR通过CprM654检测到91例（75.2%），通过CprM511检测到72例（59.5%），通过Env641检测到74例（61.1%）。在50名血清学阴性者中，通过CprM654检测到10例（20.0%），通过CprM511检测到12例（24.0%），通过Env641检测到11例（22.0%）。在血清学阳性样本中，依次使用三种方法的总体检测率为82.6%（100/121），在血清学阴性样本中为40.0%（20/50）；6.6%（8/120）的样本同时感染了多种DENV血清型。我们得出结论，针对654bp C-prM区域的改良RT-PCR方法提高了急性登革热的检测率，依次使用所有三种方法可进一步提高检测率。
