Proteinase K treatment in absence of RNA isolation classical procedures is a quick and cheaper alternative for SARS-CoV-2 molecular detection

Abstract

The World Health Organization (WHO) has declared a pandemic of COVID-19, the disease caused by the recently described SARS-CoV-2. The relevance and importance of mass diagnosis in order to find the asymptomatic individuals is widely recognized as a mandatory tool to reinforce the control measures for monitoring virus circulation and reduce the spreading of SARS-CoV-2. Here, we described quickness and cheaper strategies of direct RT-qPCR (in the absence of RNA isolation) and compared the results to those obtained using standard RNA isolation procedure. The tests varied using pure, diluted samples, combined with Proteinase K (PK) or Lysis Buffer. Our findings showed consistently that PK pre-treated samples in the absence of RNA extraction procedures presents similar results to those obtained by standard RNA isolation procedures. On average, 16 samples extracted with the MagMAX™ CORE Kit, take around 2 hours, costing an average of USD 5, the pre-treatment of samples using PK, on the other hand, would cut the value to less than USD 0.30 and reduce the time of procedure in more than 1 ½ hours. The present study suggests the use of PK treatment instead of RNA isolation in order to reduce costs and time in processing samples for molecular diagnosis of SARS-CoV-2.

RNA-extraction-free diagnostic method to detect SARS-CoV-2: an assessment from two states, India

“…Heamoptysis 1 (0. Pretreatment of such buffer eluates with proteinase K followed by heat inactivation was found to improve sensitivity in a pilot laboratory assay of SARS-CoV-2 [24], consistent with the previous reports from other researchers [13]. We conducted the current assessment to evaluate a similar approach of direct extraction from dry swabs using TE buffer and proteinase K followed by heat inactivation from nasopharyngeal specimens collected from consecutive clinic attendees.…”

“…Complete inactivation of SARS-CoV-2 was observed only after heat treatment at 95 °C for 1 or 5 min in another study [27]. On the contrary, Mallmann et al [24] tested different conditions and reported that pretreatment with proteinase K and heat treatment at 98 °C yielded best results with Ct values similar to that in a standard method; conditions similar to those followed in the current investigation.…”

“…On the contrary, an earlier evaluation of a direct extraction method using buffer eluates of the swabs transported in dry tube (without transport medium) and heat treatment at 98 o C for 6 minutes against the reference method on 978 clinical samples yielded an overall sensitivity of 56 % (95% CI 49.8 % to 61.6 %) and specificity of 95 https://doi.org/10.1017/S0950268821002302 % (95% CI 93.4 % to 96.8 %) [20]. Pretreatment of such buffer eluates with Proteinase K followed by heat inactivation was found to improve sensitivity in a pilot laboratory assay of SARS-CoV-2 [24], consistent with the previous reports from other researchers [13].…”

Natural spring water gargle samples as an alternative to nasopharyngeal swabs for SARS‐CoV‐2 detection using a laboratory‐developed test

“…Another possibility would be the use of proteinase K (PK) in the CHUQ protocol. Indeed, some studies suggested that pretreatment of samples with PK increased the sensitivity of direct rRT‐PCR for SARS‐CoV‐2 detection, 28,29 particularly in low positive samples with high C t values 13 . It has been suggested that PK could degrade or inactivate RNAses, allowing to purify a better quality of RNA 30 .…”

RNA-extraction-free diagnostic method to detect SARS-CoV-2: an assessment from two States, India

“…On the contrary, an earlier evaluation of a direct extraction method using buffer eluates of the swabs transported in dry tube (without transport medium) and heat treatment at 98 o C for 6 minutes against the reference method on 978 clinical samples yielded an overall sensitivity of 56 % (95% CI 49.8 % to 61.6 %) and specificity of 95 % (95% CI 93.4 % to 96.8 %) [20]. Pretreatment of such buffer eluates with Proteinase K followed by heat inactivation was found to improve sensitivity in a pilot laboratory assay of SARS-CoV-2 [24], consistent with the previous reports from other researchers [13].…”