Assessing the integrity of SARS-CoV-2 and F-specific RNA bacteriophage RNA in raw wastewater (ANRS0160)

RNA integrity is an essential parameter for analyzing the nature of viral particles, especially in environmental samples where assessing virus infectivity is often difficult or impossible. It is also an important factor in the effectiveness of virus sequencing in environmental matrices containing mixed viral populations composed of variants that differ from one another by only a limited number of mutations, such as in the case of SARS-CoV-2. This study introduces a multiplex Reverse Transcription Digital PCR (RT-dPCR) method for evaluating the RNA integrity of SARS-CoV-2 and F-specific RNA phages belonging to subgroup I (FRNAPH-I) using synthetic RNA, viral stocks, and then raw wastewater (WW) in which SARS-CoV-2 and FRNAPH-I were naturally present. An initial approach using one-step multiplex digital Reverse Transcription PCR (dRT-PCR) demonstrated unequal detection across the genomic regions of both FRNAPH and SARS-CoV-2. To overcome this methodological bias, a two-step method called Long-Range Reverse Transcription digital PCR (LR-RT-dPCR) was developed. This approach involves performing long-range reverse transcription at the 3' end using a single specific reverse primer to generate contiguous cDNA that spans multiple targets of interest. Following cDNA synthesis, the sample is partitioned, and a multiplex amplification is carried out on targets located at the 3' end, middle, and 5' end of the sequence. The LR-RT-dPCR method enabled uniform detection with enhanced sensitivity and was validated using capillary electrophoresis on synthetic RNA of MS2, a phage which belongs to the FRNAPH-I subgroup. LR-RT-dPCR was employed in both triplex and quintuplex formats to analyze the MS2 phage genome (3,569 nucleotides (nt)) and SARS-CoV-2 genome (∼30,000 nt), respectively. Using this approach, viral RNA integrity was evaluated through the detection frequencies of genome fragments of the whole genome. The viral stocks of MS2 phages replicated in a laboratory and stored in phosphate-buffered saline (PBS) exhibited high RNA detection frequencies (> 50 %). In WW, RNA detection frequency was significantly lower, not exceeding 2 % even for the shortest fragment of the FRNAPH-I genome. On the other hand, SARS-CoV-2 RNA showed greater detection frequency than FRNAPH-I RNA in WW, with values exceeding 30 % for short fragments (<1,500 nt) and ranging from 0 % to 44 % for longer fragments (1,500 to 3,500 nt). The relationship between the detection frequency of a fragment and its length does not appear to be strictly linear, as factors other than length can influence genome integrity. These factors include the intrinsic properties of specific genomic regions. For example, the S3-ORF3a region of the SARS-CoV-2 genome appears particularly stable.