Detection and quantification of hepatitis E virus and other foodborne viruses in food and clinical matrices – utilization of home-made MS2 phage-like particles

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00027162%3A_____%2F18%3AN0000222" target="_blank" >RIV/00027162:_____/18:N0000222 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.visavet.es/data/congresos/hepatitis-e-2018/HEV2018_Hepatitis_E_Workshop_Abstract_Book_01.pdf" target="_blank" >https://www.visavet.es/data/congresos/hepatitis-e-2018/HEV2018_Hepatitis_E_Workshop_Abstract_Book_01.pdf</a>

DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Detection and quantification of hepatitis E virus and other foodborne viruses in food and clinical matrices – utilization of home-made MS2 phage-like particles

Popis výsledku v původním jazyce

Hepatitis E: Paradigm of a food-borne zoonotic emerging disease in Europe, 4-5.6.2018. Madrid, Spain – poster. The detection and quantification of foodborne viruses in food and clinical matrices represents relatively complex, multi-step analysis which is most often ended by detection and quantification of these viruses by the real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). To identify false negative results and to guarantee the validity and reliability of obtained results it is necessary to strictly control the entire analytical process. Therefore an external control spanning/including all analytical procedures should be used for this purpose. According to ISO technical specification (ISO/TS) in the case of RT-qPCR detection of hepatitis A virus (HAV) and noroviruses (NoV), such a control is characterised as non-pathogenic virus or virus-like particle with structural properties close to those of pathogenic virus (non-enveloped, +ssRNA genome), which should not be expected to occur naturally in matrices under test while being genetically distinct from the pathogenic virus in order to be able to be reliably differentiated from it by molecular biological methods. The ISO/TS recommend using a mutant strain of mengovirus (vMC0) for this purpose. The external control of analysis fulfilling these criteria was prepared in the form of MS2 phage-like particles (MS2 PLP), which were derived from bacteriophage MS2. The MS2 PLP were compared to vMC0. The prepared MS2 PLP carry the unique control de novo-synthesized +ssRNA sequence within the capsid whose natural occurrence in the analysed sample is extremely unlikely and can therefore be used as the universal external control of analysis in RT-qPCR detection and quantification of non-enveloped +ssRNA foodborne viruses including hepatitis E virus (HEV). Since there is no ISO/TS for detection of HEV so far, the use of external control of analysis in detection of HEV is essential as well as in the detection of HAV and NoV according to existing ISO/TS. MS2 PLP were used to accurately determine the efficiency of the whole analytical process for different types of matrices. Moreover, the accurate value of the efficiency of the whole analytical process enabled to determine the HEV viral load in each analysed sample. In addition to this use, our improved system for production of MS2 PLP can be simply modified to produce MS2 PLP carrying other specific +ssRNA molecules e.g. target sequences of established RT-qPCR detection methods. MS2 PLP are extremely pure without the presence of contaminating DNA molecules and are produced in total quantity up to 1014 of MS2 PLP in the single production batch. Therefore, MS2 PLP can be utilized not only as an external control of analysis, but can also be used as a standard and/or positive control in established RT-qPCR detection methods or as a calibrator in optimizing detection methods of foodborne +ssRNA viruses in different matrices.

Název v anglickém jazyce

Detection and quantification of hepatitis E virus and other foodborne viruses in food and clinical matrices – utilization of home-made MS2 phage-like particles

Popis výsledku anglicky

Hepatitis E: Paradigm of a food-borne zoonotic emerging disease in Europe, 4-5.6.2018. Madrid, Spain – poster. The detection and quantification of foodborne viruses in food and clinical matrices represents relatively complex, multi-step analysis which is most often ended by detection and quantification of these viruses by the real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). To identify false negative results and to guarantee the validity and reliability of obtained results it is necessary to strictly control the entire analytical process. Therefore an external control spanning/including all analytical procedures should be used for this purpose. According to ISO technical specification (ISO/TS) in the case of RT-qPCR detection of hepatitis A virus (HAV) and noroviruses (NoV), such a control is characterised as non-pathogenic virus or virus-like particle with structural properties close to those of pathogenic virus (non-enveloped, +ssRNA genome), which should not be expected to occur naturally in matrices under test while being genetically distinct from the pathogenic virus in order to be able to be reliably differentiated from it by molecular biological methods. The ISO/TS recommend using a mutant strain of mengovirus (vMC0) for this purpose. The external control of analysis fulfilling these criteria was prepared in the form of MS2 phage-like particles (MS2 PLP), which were derived from bacteriophage MS2. The MS2 PLP were compared to vMC0. The prepared MS2 PLP carry the unique control de novo-synthesized +ssRNA sequence within the capsid whose natural occurrence in the analysed sample is extremely unlikely and can therefore be used as the universal external control of analysis in RT-qPCR detection and quantification of non-enveloped +ssRNA foodborne viruses including hepatitis E virus (HEV). Since there is no ISO/TS for detection of HEV so far, the use of external control of analysis in detection of HEV is essential as well as in the detection of HAV and NoV according to existing ISO/TS. MS2 PLP were used to accurately determine the efficiency of the whole analytical process for different types of matrices. Moreover, the accurate value of the efficiency of the whole analytical process enabled to determine the HEV viral load in each analysed sample. In addition to this use, our improved system for production of MS2 PLP can be simply modified to produce MS2 PLP carrying other specific +ssRNA molecules e.g. target sequences of established RT-qPCR detection methods. MS2 PLP are extremely pure without the presence of contaminating DNA molecules and are produced in total quantity up to 1014 of MS2 PLP in the single production batch. Therefore, MS2 PLP can be utilized not only as an external control of analysis, but can also be used as a standard and/or positive control in established RT-qPCR detection methods or as a calibrator in optimizing detection methods of foodborne +ssRNA viruses in different matrices.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

30302 - Epidemiology

Projekt

<a href="/cs/project/NV17-31921A" target="_blank" >NV17-31921A: Viry v souvislosti s alimentárními infekcemi – molekulární epidemiologie a metody rychlé detekce</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2018

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů