The mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and can establish host-specific lineages. The four main hosts are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV matrix protein (MP)-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93-97% and LOD95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.
- MeSH
- chřipka lidská diagnóza virologie MeSH
- infekce viry z čeledi Orthomyxoviridae diagnóza virologie MeSH
- lidé MeSH
- nemoci prasat diagnóza virologie MeSH
- One Health MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- prasata MeSH
- ptačí chřipka u ptáků diagnóza virologie MeSH
- ptáci virologie MeSH
- reprodukovatelnost výsledků MeSH
- virus chřipky A, podtyp H1N1 genetika izolace a purifikace MeSH
- virus chřipky A, podtyp H3N2 genetika izolace a purifikace MeSH
- virus chřipky A genetika izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
The ongoing evolution of microbial pathogens represents a significant issue in diagnostic PCR/qPCR. Many assays are burdened with false negativity due to mispriming and/or probe-binding failures. Therefore, PCR/qPCR assays used in the laboratory should be periodically re-assessed in silico on public sequences to evaluate the ability to detect actually circulating strains and to infer potentially escaping variants. In the work presented we re-assessed a RT-qPCR assay for the universal detection of influenza A (IA) viruses currently recommended by the European Union Reference Laboratory for Avian Influenza. To this end, the primers and probe sequences were challenged against more than 99,000 M-segment sequences in five data pools. To streamline this process, we developed a simple algorithm called the SequenceTracer designed for alignment stratification, compression, and personal sequence subset selection and also demonstrated its utility. The re-assessment confirmed the high inclusivity of the assay for the detection of avian, swine and human pandemic H1N1 IA viruses. On the other hand, the analysis identified human H3N2 strains with a critical probe-interfering mutation circulating since 2010, albeit with a significantly fluctuating proportion. Minor variations located in the forward and reverse primers identified in the avian and swine data were also considered.
Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates.
- MeSH
- barvení a značení * MeSH
- DNA sondy metabolismus MeSH
- fluorescence MeSH
- hydrolýza MeSH
- kalibrace MeSH
- koně MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- nukleové kyseliny metabolismus MeSH
- psi MeSH
- reprodukovatelnost výsledků MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
