MutaPLEX® ENTEROVIRUS Real Time RT-PCR KIT
INTENDED USE
The MutaPLEX® Enterovirus real time RT-PCR kit is a qualitative assay for the specific detection of enterovirus (Polio-, Coxsackie A-, Coxsackie B- and Echoviruses) RNA in stool or other clinical sample matrices (whole blood, plasma, respiratory samples, CSF [cerebrospinal fluid] etc.) using open real time RT-PCR systems (e.g. from ABI, Stratagene, Corbett Research: RotorGene, Cepheid: Smart Cycler, or Light Cycler 480 (Roche). In parallel, the RNA extraction efficiency from stool samples is controlled by a separate internal control.
INTRODUCTION
Human enteroviruses are ubiquitous high-infectious pathogens belonging to the family of Picornavirida. They have a high incidence worldwide (about 500 million infections/ year). The non-enveloped viruses (with ss-RNA genome of 6-7 kb) are high resistant even against pH values from 3-9. Thus far, there is no effective antiviral chemotherapeutics available. Until now about 70 serotypes are known. Route of transmission in human is fecal-oral by contact and smear infection. Contaminated food and water (also knifes and forkes) are an important source of infection for this highly contagious viruses which can be secreted with stool from infected patients for several weeks.
Enteroviruses may trigger many (even life-threatening) infections, especially among children. In summer period very often contaminated waters (swimming pools, lakes) change for the worse. Enteroviruses cause several diseases, e.g. infection of upper respiratory tract, summer flue, herpangina, hand-foot-mouth-disease, juvenile diabetes mellitus, infection of gastrointestinal tract, eye-infections or epidemic myalgie. But also myocarditis, paralysis, multiple organ failure, meningitis and encephalitis may be associated with enterovirus infections.
PRINCIPLE OF THE TEST
MutaPLEX® Enterovirus real time RT-PCR kit
MutaPLEX® Enterovirus real time RT-PCR kit contains specific primers, fluorescence-marked probes and additional material for the detection of enterovirus RNA in clinical samples. Human stool or other clinical samples, such as whole blood, plasma, respiratory samples, CSF (cerebrospinal fluid) etc. can be used as starting material for RNA-extraction and following pathogen analysis with MutaLPLEX® Enterovirus real time RT-PCR kit.
Reverse transcription (RT) of potentially contained viral RNA to cDNA and subsequent amplification of enterovirus-specifc fragments by means of PCR is done in only one step. Target sequence for the detection is within the 5`-untranslated region (5`-UTR) of the genome. Detection of enterovirus amplificates is achieved in real time by hybridization and subsequent hydrolysis of enterovirus-specific fluorescence probes. Their emitted signal is measured (during PCR process) by the optical unit of the real time PCR system in use (ABI: PRISM SDS-, Stratagene: MxPRO-, Corbett Research: RotorGene 3000/ 6000-, Cepheid: SmartCycler- Software, Roche: LC480- software). Enterovirus-specific amplification is measured in FAMchannel (470/ 510 nm).
Furthermore, MutaPLEX® Enterovirus real time RT-PCR - kit contains also a separate RNA extraction control. This is added initially prior to the nucleic acid extraction procedure and detected in the follow-up by an independent amplification step. This internal control (IC) allows on the one hand the exposure of errors during RNA extraction procedure and identifies on the other hand a potential inhibition of reverse transcription or PCR. Doing this, the risk for falsenegative results is minimized extensively. RNA extraction control (= internal control for PCR) is measured in VIC- (respectively = HEX/ JOE/ TET/ Cy3) channel (530/ 555 nm).
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