Heat inactivation decreases the qualitative real-time RT-PCR detection rates of clinical samples with high cycle threshold values in COVID-19

Zou, Jialin; Zhi, Shenshen; Su, Xingyu; Lee, Kang; Li, Caiyu; Su, Xiaosong; Zhang, Shiyin; Ge, Shuping; Li, Wei

doi:10.1016/j.diagmicrobio.2020.115109

Cited by 32 publications

(35 citation statements)

References 7 publications

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“…In this study the Ct value was not significantly affected by heating to 56 °C and 60 °C. This is in agreement with some studies ( Pastorino et al, 2020 ; Wang et al, 2020b ) but not others ( Pan et al, 2020 ; Zou et al, 2020 ). We also found that heating to 80 °C for 30 min or more led to an increase in Ct value and therefore a reduction in RT-PCR sensitivity that could impact upon clinical diagnosis.…”

Section: Discussionsupporting

confidence: 94%

The effect of heat-treatment on SARS-CoV-2 viability and detection

Burton

Love

Richards

et al. 2021

Journal of Virological Methods

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Section: Discussionsupporting

confidence: 94%

The effect of heat-treatment on SARS-CoV-2 viability and detection

Burton

Love

Richards

et al. 2021

Journal of Virological Methods

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“…Integrity was not significantly affected by heating to 56°C and 60°C. This is in agreement with some studies (17, 26) but not others (27, 28). In this study we found that heating to 80°C for 30 minutes or more led to an increase in Ct value and therefore a reduction in RT-PCR sensitivity that could impact upon clinical diagnosis.…”

Section: Discussionsupporting

confidence: 94%

The effect of heat on SARS-CoV-2 viability and RNA integrity as determined by plaque assay, virus culture and real-time RT-PCR

Burton

Love

Richards

et al. 2020

Preprint

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The effect of heat on SARS-CoV-2/England/2/2020 viability was assessed by plaque assay and virus culture. Heating to 56°C and 60°C for 15, 30 and 60 minutes led to a reduction in titre of between 2.1 and 4.9 log 10 pfu/ml but complete inactivation was not observed. At 80°C plaques were observed after 15 and 30 minutes of heating, however after 60 minutes viable virus was only detected following virus culture. Heating to 80°C for 90 minutes and 95°C for 1 and 5 minutes resulted in no viable virus being detected. At 56°C and 60°C significant variability between replicates was observed and the titre often increased with heat-treatment time. Nucleic acids were extracted and tested by RT-PCR. Sensitivity of the RT-PCR was not compromised by heating to 56°C and 60°C. Heating to 80°C for 30 minutes or more and 95°C for 1 or 5 minutes however, resulted in an increase of at least three Ct values. This increase remained constant when different dilutions of virus underwent heat treatment. This indicates that high temperature heat inactivation of clinical samples prior to nucleic acid extraction could significantly affect the ability to detect virus in clinical samples from patients with lower viral loads by RT-PCR.

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“…Indeed we avoided to use the most commonly used guanidinium to circumvent possible cause of inhibition of the amplification 24 , 25 . However, the heating step was used only for the direct quantitation and it could be another possible cause of underestimation of the amplification as reported recently 26 .…”

Section: Discussionmentioning

confidence: 99%

Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR

Deiana

Mori

Piubelli

et al. 2020

Sci Rep

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Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several viruses. The aim of this study was to evaluate the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance, comparing the direct quantitation of SARS-CoV-2 on nasopharyngeal swab with the procedure applied to the extracted RNA. Moreover, two widely used swab types were compared (UTM 3 mL and ESwab 1 mL, COPAN). A total of 50 nasopharyngeal swabs (n = 25 UTM 3 mL and n = 25 ESwab 1 mL) from SARS-CoV-2 patients, collected during the pandemic at IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy), were used for our purpose. After heat inactivation, an aliquot of swab medium was used for the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the extracted RNA. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches. In conclusion, we described a direct quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples and to the heating treatment, as we used thawed and heat inactivated material. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.

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Heat inactivation decreases the qualitative real-time RT-PCR detection rates of clinical samples with high cycle threshold values in COVID-19

Cited by 32 publications

References 7 publications

The effect of heat-treatment on SARS-CoV-2 viability and detection

The effect of heat-treatment on SARS-CoV-2 viability and detection

The effect of heat on SARS-CoV-2 viability and RNA integrity as determined by plaque assay, virus culture and real-time RT-PCR

Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR

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