Critical parameters of real time reverse transcription polymerase chain reaction (RT-PCR) diagnostics: Sensitivity and specificity for bluetongue virus

Piet A Van Rijn,Jan Boonstra

doi:10.1016/j.jviromet.2021.114211

Piet A Van Rijn, Jan Boonstra

Open Access

https://doi.org/10.1016/j.jviromet.2021.114211

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Abstract

A new variant of bluetongue virus serotype 3, BTV3 ITL 2018 (here named: BTV3), was included in serial dilutions in the BT Proficiency Test 2020. Although the OIE-recommended panBTV real time RT-PCR test targeting genome segment 10 (Seg-10) detected this variant, we showed that reverse transcription (RT) at 61 °C instead of 50 °C completely abolished detection. Another Seg-10 panBTV real time RT-PCR test detected BTV3, irrespective of the temperature of RT. In silico validation showed that each of the OIE-recommended PCR primers using IVI-primers contain single mismatches at the -3 position for BTV3. In contrast, WBVR-primers of a second test completely match to the BTV3 variant. Our results suggest that single mismatches caused false negative PCR results for BTV3 at high RT temperature. Indeed, correction of both IVI-primers for BTV3 led to positive results for BTV3 but negative results for all other samples of the BT Proficiency Test 2020. Apparently, variability of the -3 position is sufficient for discriminative PCR detection, although the single mismatch in the IVI-reverse primer was the most important for this phenomenon. Extensive in silico validation showed that targets of both Seg-10 panBTV RT-PCR tests are not completely conserved, and the detailed effect of single mismatches are hard to predict. Therefore, we recommend at least two panBTV RT-PCR tests to minimize the risk of false negatives. Preferably, their PCR targets should be located at completely different and highly conserved regions of the BTV genome to guarantee adequate detection of future BTV infections.

Highlights

A new variant of bluetongue virus serotype 3, BTV3 ITL 2018, was included in serial di lutions in the BT Proficiency Test 2020
Boonstra require denaturation of extracted viral double stranded RNA (dsRNA) before reverse transcription (RT) and polymerase chain reaction (PCR) are performed as described for many RT-PCR tests, i.e. the Organisation for Animal Health (OIE)-recommended panBTV RT-PCR test (OIE, 2019a)
To reduce labour and the risk of contamination, we have developed the all-in-one method by which all steps, predenaturation, RT, PCR, and real time detection are performed after the complete RT-PCR mix was added to isolated dsRNA

Summary

Introduction

A new variant of bluetongue virus serotype 3, BTV3 ITL 2018 (here named: BTV3), was included in serial di lutions in the BT Proficiency Test 2020. These are supposed to detect all and future BTV variants (panBTV PCR tests). In silico validation by the automated tool PCRv demonstrated that Seg-10 panBTV PCR tests target highly conserved, specific regions of Seg-10 (van Weezep et al, 2019).

Results

Conclusion