RR:C19 Evidence Scale rating by reviewer:
Potentially informative: The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.
This techniques paper entitled “Clinical validation of innovative, low cost, kit-free, RNA processing protocol for RT-PCR based COVID-19 testing” by Sahajpal et al., aims to determine if SARS-CoV2 RNA can be amplified from nasopharyngeal swab samples. To do so, they tested various conditions that used heat denaturing of swab samples with or without isopropanol step to precipitate nucleic acids. While heat denaturing (direct RT-PCR) method did not result in amplification of viral RNA for nucleocapsid region, treatment of samples at 90˚C for 5 minutes followed by isopropanol method yielded amplicons that were comparable to methods approved by the FDA-EUA. While the study has some merits, there are several shortcomings that need to be addressed.
This is a technique-focused study, yet the methods section is very short, broad and lacks any details that would allow for this technique to be replicated by others.
1. What were the buffer conditions in which the samples were suspended and subjected to heat-shock treatment? This claims to be kit-free and low cost, hence details should be provided to make buffers and reagents and a cost estimate for an entire reaction should be given.
2. Was any salt used for isopropanol precipitation? If so, which salt and would that interfere with amplification steps and if one wash with 75% ethanol was sufficient to remove all contaminants?
3. Since the authors claim that this technique has advantage over other traditional methods, how long after sample collection in the field can SARS-CoV-2 RNA can be amplified?
4. The nucleocapsid region (N) or open reading frame (ORF) were amplified- what were the primer sequence used (in two different methods) and do they cross react with other N or ORF genes of other SARS viruses?
5. How was specificity confirmed? Was the amplified product sequenced? Melting-curve analysis is not fool proof; there is no mention of a negative control.
6. Previously confirmed positive or negative cases are a good start, but no method so far is caveat-free and thus use of an internal negative control is essential. How many replicates were used?
7. What were the precise cycling conditions used? The data shown in Figures 2 and 3 that compares Ct values with one value are misleading.
8. This reviewer assumes that different primer sequences were used for amplification of either N gene or ORF1ab gene when using two different methods and thus absolute Ct value cannot be compared as reagents, primers and probes or reaction volumes involved are presumably different.