Kumar Satyendra, Jadi Ramesh S, Anakkathil Sudeep B, Tandale Babasaheb V, Mishra Akhilesh Chandra, Arankalle Vidya A
National Institute of Virology, Pashan, Pune, India.
BMC Infect Dis. 2008 Dec 17;8:168. doi: 10.1186/1471-2334-8-168.
Chandipura virus (CHPV), a member of family Rhabdoviridae was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55-75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR) using TaqMan technology was developed for rapid diagnosis.
Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [in vivo (mice) in ovo (eggs), in vitro (Vero E6, PS, RD and Sand fly cell line)] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard.
Real-time one step RT-PCR was optimized using in vitro transcribed (IVT) RNA. Standard curve showed linear relationship for wide range of 102-1010 (r2 = 0.99) with maximum Coefficient of variation (CV = 5.91%) for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice) used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 x 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 x 102 PFU/ml). Vero and PS cell-lines (1.2 x 103 PFU/ml) were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy individual was used.
On account of the high sensitivity, reproducibility and specificity, the assay can be used for the rapid detection and quantitation of CHPV RNA from clinical samples during epidemics and from endemic areas. The assay may also find application in screening of antiviral compounds, understanding of pathogenesis as well as evaluation of vaccine.
钱迪普拉病毒（CHPV）是弹状病毒科的成员，2003年在印度安得拉邦引发了儿童急性脑炎的爆发，并于2004年在印度西部古吉拉特邦的部落儿童中引发了小规模疫情。病死率在55%-75%之间。鉴于该疾病进展迅速且死亡率高，开发了一种使用TaqMan技术通过实时一步逆转录PCR（实时一步RT-PCR）对CHPV RNA进行定量的高灵敏度方法，用于快速诊断。
设计了针对P基因的引物和探针，并用于标准化CHPV RNA定量的实时一步RT-PCR检测。通过PCR扩增、TA克隆和体外转录制备标准RNA。将优化后的实时一步RT-PCR检测与诊断性巢式RT-PCR以及不同的病毒分离系统[体内（小鼠）、卵内（鸡蛋）、体外（Vero E6、PS、RD和白蛉细胞系）]用于检测CHPV。以被视为金标准的诊断性巢式RT-PCR评估实时一步RT-PCR检测的灵敏度和特异性。
使用体外转录（IVT）RNA对实时一步RT-PCR进行了优化。标准曲线在102-1010的宽范围内显示出线性关系（r2 = 0.99），IVT RNA的最大变异系数（CV = 5.91%）。新开发的实时RT-PCR在灵敏度方面与巢式RT-PCR相当，并优于用于病毒分离的细胞系和其他活体系统（鸡胚和幼鼠）。实时一步RT-PCR和巢式RT-PCR的检测限为1.2 x 100 PFU/ml。RD细胞、白蛉细胞、幼鼠和鸡胚显示出几乎相同的灵敏度（1.2 x 102 PFU/ml）。Vero和PS细胞系（1.2 x 103 PFU/ml）对CHPV感染最不敏感。当使用来自其他病毒或健康个体的RNA时，该检测的特异性为100%。
由于具有高灵敏度、可重复性和特异性，该检测可用于在疫情期间从临床样本以及从流行地区快速检测和定量CHPV RNA。该检测还可用于筛选抗病毒化合物、了解发病机制以及评估疫苗。
