Critical parameters of real time reverse transcription polymerase chain reaction (RT-PCR) diagnostics: sensitivity and specificity for bluetongue virus
A brand new variant of bluetongue virus serotype 3, BTV3 ITL 2018 (right here named: BTV3), was included in serial dilutions within the BT Proficiency Take a look at 2020. Though the OIE-recommended panBTV actual time RT-PCR take a look at focusing on genome section 10 (Seg-10) detected this variant, we confirmed that reverse transcription (RT) at 61 °C as an alternative of 50 °C utterly abolished detection. One other Seg-10 panBTV actual time RT-PCR take a look at detected BTV3, regardless of the temperature of RT.
In silico validation confirmed that every of the OIE-recommended PCR primers utilizing IVI-primers include single mismatches on the -Three place for BTV3. In distinction, WBVR-primers of a second take a look at utterly match to the BTV3 variant. Our outcomes recommend that single mismatches induced false unfavorable PCR outcomes for BTV3 at excessive RT temperature.
Certainly, correction of each IVI-primers for BTV3 led to constructive outcomes for BTV3 however unfavorable outcomes for all different samples of the BT Proficiency Take a look at 2020. Apparently, variability of the -Three place is adequate for discriminative PCR detection, though the only mismatch within the IVI-reverse primer was a very powerful for this phenomenon.
In depth in silico validation confirmed that targets of each Seg-10 panBTV RT-PCR exams are usually not utterly conserved, and the detailed impact of single mismatches are onerous to foretell. Subsequently, we advocate at the least two panBTV RT-PCR exams to attenuate the danger of false negatives. Ideally, their PCR targets needs to be situated at utterly completely different and extremely conserved areas of the BTV genome to ensure ample detection of future BTV infections.
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