Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of Chikungunya virus

Santhosh, S.R.; Parida, Manmohan; Dash, Paban Kumar; Pateriya, Atul Kumar; Pattnaik, Bramhadev; Pradhan, H.K.; Tripathi, Nagesh K.; Ambuj, S.; Gupta, Nimesh; Saxena, Parag; Rao, P. Venkata

doi:10.1016/j.jcv.2007.04.015

Cited by 69 publications

(48 citation statements)

References 13 publications

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“…The molecular diagnosis of CHIKV has only been evaluated for human samples [16][17][18][19][20][21][22][23] and no real-time RT-PCR assay exists for detecting ONNV present in human or mosquito F igure 1. Detection of CHIKV in infected mosquitoes using real-time reverse transcription-polymerase chain reaction.…”

Section: Discussionmentioning

confidence: 99%

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Development of Field-Based Real-Time Reverse Transcription–Polymerase Chain Reaction Assays for Detection of Chikungunya and O’nyong-nyong Viruses in Mosquitoes

et al. 2009

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Abstract. Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaqueforming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.

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Section: Discussionmentioning

confidence: 99%

“…Several groups have developed real-time RT-PCR assays for the molecular diagnosis of CHIK involving human samples. [16][17][18][19][20][21][22][23] However, these assays have not been evaluated for detecting CHIKV RNA in infected mosquitoes. Additionally, no real-time RT-PCR assay exists for detecting ONNV RNA in human or mosquito samples.…”

Section: Introductionmentioning

confidence: 99%

Development of Field-Based Real-Time Reverse Transcription–Polymerase Chain Reaction Assays for Detection of Chikungunya and O’nyong-nyong Viruses in Mosquitoes

et al. 2009

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“…6) and was used for rapid screening of CHIKV suspected samples in Andhra Pradesh, India as well as Southern Thailand [41,80]. A real time RT-PCR test has been developed for CHIKV diagnosis [19,50,63]. RT-LAMP assay is highly sensitive and costeffective tool, and can be used effectively during disease surveillance [48].…”

Section: Diagnosismentioning

confidence: 99%

Reemergence of Chikungunya virus in Indian Subcontinent

Kumar

Gopal

2010

Indian J. Virol.

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Chikungunya virus (CHIKV), a reemerging arboviral disease of public health concern is characterized by a triad of fever, rash and arthralgia. It was responsible for a number of epidemics in Asia and Africa. The severity of the current epidemic can be judged by the fact that an estimated 1.38 million people in India and one-third of the La Reunion population (by April 2006) were affected by CHIKV. Aedes aegypti and Aedes albopictus are the major mosquitoes transmitting CHIKV in Asia. Various neurological complications and CHIKV associated deaths were encountered during the current outbreak (2005)(2006)(2007)(2008)(2009)(2010). The aggressive nature of the recent CHIKV epidemic was attributed to the mutations in the viral genome in addition to their adaptation and spread to vectors like Aedes albopictus. Proper diet, adequate rest and symptomatic treatment using non-salicylate analgesics and Non-steroidal anti inflammatory drugs (NSAIDS) helped the patients in recovering from CHIKV infections. In the absence of an effective vaccine, rapid implementation of mosquito control measures and establishment of a system for continuous surveillance of the disease seems to be the only possible solution to prevent any such outbreak in the near future.

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“…Several groups have also developed real-time RT-PCR assays for the detection and quantification of CHIKV in clinical samples [13,14]. However, compared to conventional RT-PCR, these methods are expensive, require sophisticated high precision instrument and are restricted to reference laboratory with good financial and technical resources.…”

Section: Introductionmentioning

confidence: 99%

Development of a Quantitative Competitive Reverse Transcription Polymerase Chain Reaction (QC-RT–PCR) for Detection and Quantitation of Chikungunya Virus

et al. 2010

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Chikungunya is one of the most important emerging arboviral infections of public health significance. Due to lack of a licensed vaccine, rapid diagnosis plays an important role in early management of patients. In this study, a QC-RT-PCR assay was developed to quantify Chikungunya virus (CHIKV) RNA by targeting the conserved region of E1 gene. A competitor molecule containing an internal insertion was generated, which provided a stringent control of the quantification process. The introduction of 10-fold serially diluted competitor in each reaction was further used to determine sensitivity. The applicability of this assay for quantification of CHIKV RNA was evaluated with human clinical samples, and the results were compared with real-time quantitative RT-PCR. The sensitivity of this assay was estimated to be 100 RNA copies per reaction with a dynamic detection range of 10(2) to 10(10) copies. Specificity was confirmed using closely related alpha and flaviviruses. The comparison of QC-RT-PCR result with real-time RT-PCR revealed 100% concordance for the detection of CHIKV in clinical samples. These findings demonstrated that the reported assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of CHIKV in acute-phase serum samples.

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Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of Chikungunya virus

Cited by 69 publications

References 13 publications

Development of Field-Based Real-Time Reverse Transcription–Polymerase Chain Reaction Assays for Detection of Chikungunya and O’nyong-nyong Viruses in Mosquitoes

Development of Field-Based Real-Time Reverse Transcription–Polymerase Chain Reaction Assays for Detection of Chikungunya and O’nyong-nyong Viruses in Mosquitoes

Reemergence of Chikungunya virus in Indian Subcontinent

Development of a Quantitative Competitive Reverse Transcription Polymerase Chain Reaction (QC-RT–PCR) for Detection and Quantitation of Chikungunya Virus

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