{"id":2881,"date":"2021-11-17T18:14:13","date_gmt":"2021-11-17T17:14:13","guid":{"rendered":"https:\/\/www.oncology-and-cytogenetic-products.com\/rt-pcr-the-gold-standard-in-covid-19-diagnosis-the-most-common-pitfalls-and-their-interpretations\/"},"modified":"2021-11-17T18:14:14","modified_gmt":"2021-11-17T17:14:14","slug":"rt-pcr-the-gold-standard-in-covid-19-diagnosis-the-most-common-pitfalls-and-their-interpretations","status":"publish","type":"post","link":"https:\/\/www.oncology-and-cytogenetic-products.com\/it\/rt-pcr-the-gold-standard-in-covid-19-diagnosis-the-most-common-pitfalls-and-their-interpretations\/","title":{"rendered":"RT- PCR, the gold standard in CoViD-19 diagnosis: the most common pitfalls and their interpretations"},"content":{"rendered":"

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The gold standard<\/em> for SARS-CoV-2 diagnosis is the Reverse Transcriptase Polymerase Chain Reaction<\/strong>, aka RT-PCR or qRT-PCR.<\/span><\/h4>\n

This technique has been introduced in 1983 (Saiki et al. 1985) and nowadays is widely used in the diagnosis of many pathologies, from infectious diseases to metabolic, oncologic, and degenerative<\/strong> (Valones et al. 2009).<\/span><\/h4>\n

RT-PCR has been indicated as the first choice in the diagnosis of SARS-CoV-2<\/strong> and it is used with a standard protocol suggested by American CDC<\/em> (https:\/\/www.fda.gov\/media\/134922\/download).<\/span>
<\/strong><\/h4>\n

<\/strong><\/h3>\n

Extraction<\/strong><\/h3>\n

This technique begins after RNA is extracted from infected (or presumed infected subjects) cells by the use of a dedicated swab (Corman et al. 2020).<\/p>\n

Amplification<\/strong><\/h3>\n

RT-PCR copies many and many times the viral genome, if present, allowing to obtain a relative quantification of the viral load.<\/p>\n

The copy phase, called amplification<\/em>, is needed to make viral sequence quantifiable thanks to specific adapters (or primers), synthesized ad hoc, able to recognize just the viral genes directed to (Zou et al. 2020). The specificity of the target genes makes it possible to discriminate the presence of SARS CoV-2 virus from all the others, even if it belongs to the same Corona<\/em> family: the absence of those genes does not allow the copy machinery which, overall, restarts 45 times (cycles) (Corman et al. 2020). Each cycle is characterized by several steps: recognizing phase (denaturation<\/em> and annealing<\/em>) of the primers to the target, copy (elongation<\/em>) of the target sequence, and gene detection<\/em>.<\/p>\n

The amplicon is detected through a specific fluorescence probe: each probe, specific for each target amplified, is associated with a unique fluorophore.<\/p>\n

Positive and negative controls<\/strong><\/h3>\n

Positive and negative controls are performed at the same time on the unknow<\/em>n samples: it is the comparison between the results on the unknown and the controls that defines the good quality of the tests.<\/p>\n

Moreover, the controls are needed to set the threshold up<\/strong>.<\/p>\n

The threshold line must be settled just immediately upper the amplification line of the backgrounds of controls. The threshold must intersect the exponential phase of individual amplification plots<\/strong> \u00a0In the cases when the Ct<\/sub>\u00a0values are very high and the \u0394Rn<\/sub>\u00a0values are very low, the exponential phase of the amplification plot will not be well defined, so it will be necessary to make threshold adjustments that generate a (qualitative) positive sample (Bustin and Nolan 2004).<\/p>\n

What could go wrong: pitfalls in the pre-analytical and analytical phase<\/strong><\/h3>\n

As diagnosis with RT-PCR is a multistage process, it\u2019s likely to encounter errors that affect the result.<\/p>\n

Two main stages can be identified: pre-analytical phase<\/em><\/strong> (reagents chose, sample collection, conservation, RNA extraction, and its conservation) and analytical phase<\/em><\/strong> (RT-PCR).<\/p>\n

Pre-analytical phase pitfalls<\/h4>\n

Most pre-analytical<\/u><\/em><\/strong> errors are often associated with the use of:<\/p>\n