Usefulness of the extraction-free RT-PCR methods for the SARS-CoV-2 diagnostics: An Italian experience

The extraction-based real-time reverse transcription polymerase chain reaction (RT-PCR) is currently the “gold standard” for the SARS-CoV-2 diagnostics. However, some extraction-free RT-PCR techniques have been recently developed. In this study we compared the performance of heated and unheated extraction-free methods with the traditional extraction-based SARS-CoV-2 RT-PCR. The unheated extraction-free showed a perfect agreement with the standard extraction-based RT-PCR. By contrast, the heat-treated technique was associated with an 8.2% false negativity rate. The unheated extraction-free RT-PCR for the SARS-CoV-2 molecular diagnostics is a valuable alternative to the traditional extraction-based methods and may accelerate turnaround times by about two hours.

Nowadays, the real-time reverse transcription polymerase chain reaction (RT-PCR) is considered the "gold standard" assay for diagnosis of both symptomatic and asymptomatic cases [6] and included in the protocols issued by several international institutional bodies [7,8]. These commonly used protocols have a ribonucleic acid (RNA) extraction step that may be seen as an important bottleneck in the routine laboratory testing process [9].
Since March 2020, some manufacturers reported supply shortages for RNA extraction kits driven by the sudden increase in their global demand. This situation called for alternative protocols with similarly high diagnostic accuracy in order to ensure the continuity of testing [10][11][12][13]. Consequently, different direct approaches that avoid RNA extraction have been suggested, including heat-processed methods [9,14].
The objective of this study was to evaluate the diagnostic performance of two different extraction-free RT-PCR techniques and to compare their performance to the traditional extraction-based method in the realworld setting.

Methods
In this study a total of 98 con rmed nasopharyngeal swabs from COVID-19 symptomatic individuals were analysed. These samples were routinely collected in October 2020 and were eluted in the universal transport medium (UTM™, Copan Diagnostics Inc, US) and processed at the regional reference laboratory for COVID-19 diagnostic of the San Martino Policlinico Hospital, Hygiene Unit, Genoa, Liguria, Northwest Italy.
All samples were processed in parallel by using three methods: (i) standard extraction-based (EB) method performed with STARMag 96x4 Viral DNA/RNA 200C Kit (Seegene Inc., South Korea); (ii) RNA extractionfree (EF) method with a heating step (EFh+) and (iii) unheated RNA extraction-free technique (EFh-). For the EB method, total RNA was extracted and set up for RT-PCR by means of the Nimbus IVD Seegene platform using the Allplex™ 2019-nCoV Assay kit (Seegene Inc., South Korea), according to the manufacturer's instructions. For the EFh + method, thermolysis was rst performed at 95°C for 5 min and at 55°C for 3 min on Biorad CFX96™ thermal cycler (Bio-Rad Laboratories, US). EFh-method was identical to EFh + unless the thermolysis step.
The material thus obtained was tested for the identi cation of SARS-CoV-2 by means of a one-step realtime multiplex RT-PCR quantitative assay on Biorad CFX96™ thermal cycler, targeting the nucleoprotein region (N), RNA-dependent RNA-polymerase (RdRp)/spike (S) proteins and envelop (E) region. Samples showing a cycle threshold (Ct) value < 40 for at least two genes were considered positive.
The standard EB technique was considered a reference method against both EFh + and EFh-. A sensitivity with 95% con dence intervals (CIs) was calculated. As the observed Ct values were approximately normally distributed, the average target-speci c difference in Ct values between the three techniques was computed and compared by applying the repeated measures analysis of variance (ANOVA) and follow-up Tukey honestly signi cant difference post-hoc test. Data were analysed in R stats packages, v. 4.0.3 [15].

Results
On considering the traditional EB technique as a "gold standard", EFh-method displayed a perfect accordance with no false negative results [sensitivity of 100% (95% CI: 95.3-100%)]. By contrast, a total of eight (8.2%) swabs treated as per EFh + methodology were deemed false negative, giving a sensitivity of 91.8% (95% CI: 84.1-96.2%). Table 1 report the summary distributions and mean differences of Ct values according to the technique used and gene target. The omnibus ANOVA test rejected (P < 0.001) the null hypothesis that Ct values gured out by the three methods used had identical means; moreover, all the pairwise post-hoc comparisons were also statistically signi cant (P < 0.01). Indeed, the average Ct value for any gene was the lowest in EB group and the highest in the EFh+. Moreover, the highest between-method difference concerned RdRp/S gene, while the lowest one N gene (Table 1). Of note, all the eight false negatives determined by EFh + method had a Ct value > 30, independently from gene target.

Declarations
Availability of data and materials The whole raw dataset used may be available from the corresponding author on reasonable request and following approval from the San Martino Policlinico Hospital

Funding
This study received no external funding

Ethics statement
The study was conducted according to the guidelines of the Declaration of Helsinki. Ethical review and approval were waived for this retrospective analysis because it was based on the routine COVID-19 testing Figure 1 Distribution of the cycle threshold values by method used and gene target