Rapid and Real-time Assays for Detection and Quantification of Chikungunya Virus

MM Parida; SR Santhosh; PK Dash; PV Lakshmana Rao

Disclosures

Future Virology. 2008;3(2):179-192. 

In This Article

PCR-based Genomic Detection

Recently, a RT-PCR technique for diagnosing CHIKV has been developed using primer pairs amplifying specific components of three structural gene regions, Capsid (C), Envelope E1 and E2, and part of nonstructural protein (NSP)1.[18,19] To date, only conventional RT-PCR methods have been suggested for the study of CHIKV replication in supernatants, clinical samples or for epidemiological survey. In 2002, Hasebe et al. first reported a rapid, sensitive and virus-specific RT-PCR assay based on E1 and NSP1 gene targets for both quick detection and genotyping of CHIKV, especially in dengue epidemic areas. The sensitivity of this reported RT-PCR system was 5-50 plaque-forming units (PFUs; corresponding to ~500-5000 copies).[18] Subsequently in the same year, Pfeiffer et al. developed a combination of RT-PCR and nested PCR for the specific detection of CHIKV RNA. In the first step, a 427-bp fragment of the E2 gene was amplified by RT-PCR and subsequent second round amplification was performed to further enhance sensitivity and specificity. This RT-PCR/nested PCR combination was able to amplify a CHIKV-specific 172-bp amplicon from a sample containing as few as ten genome equivalents.[20,21] This assay was successfully applied to four CHIKV isolates from Asia and Africa as well as to a vaccine strain developed by The United States Army Medical Research Institute for Infectious Diseases (USAMRIID). A RT-PCR with Alphavirus-specific primers followed by multiplex nested PCR employing species-specific primers was reported for the rapid detection and identification of 14 Brazilian alphaviruses and was found to be 1000-fold more sensitive as compared with single step RT-PCR.[22]

The existing RT-PCR test systems are time consuming and labor-intensive, with a very high risk of contamination primarily due to post-PCR handling leading to carry over. In addition, the majority of diagnostic PCR assays reported to date have been used in a qualitative, or "yes/no" format. Therefore a rapid, specific and sensitive test is necessary for effective surveillance of new CHIKV circulating strains. Moreover, no quantitative molecular tool has been described to study CHIKV replication or detection in clinical samples and cell culture supernatants. However, it is important to identify and quantify this infection for epidemiological studies. In fact, viral load should be a useful marker of disease progression and a measure of antiviral compound efficiency. Increased surveillance in tropical and subtropical areas is also necessary to understand the emergence of this new disease in previously unaffected regions.

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