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. 2022 Feb 21;22(1):163.
doi: 10.1186/s12879-022-07134-7.

Effect of heat inactivation for the detection of severe acute respiratory syndrome-corona virus-2 (SARS-CoV-2) with reverse transcription real time polymerase chain reaction (rRT-PCR): evidence from Ethiopian study

Belete Woldesemayat  1 , Gebremedihin Gebremicael  2 , Kidist Zealiyas  2 , Amelework Yilma  2 , Sisay Adane  2 , Mengistu Yimer  2 , Gadissa Gutema  2 , Altaye Feleke  2 , Kassu Desta  3
Affiliations

Effect of heat inactivation for the detection of severe acute respiratory syndrome-corona virus-2 (SARS-CoV-2) with reverse transcription real time polymerase chain reaction (rRT-PCR): evidence from Ethiopian study

Belete Woldesemayat et al. BMC Infect Dis. .

Abstract

Background: Coronavirus disease 2019 (COVID-19) has been a major public health importance and its specimen needs to be handled safely due to concerns of potential transmissibility to health care workers. Heat inactivation of the sample before nucleic acid isolation might permit safe testing processes. Hence, it is important to assess the effect of heat inactivation on SARS-CoV-2 RT-PCR detection in resource limited settings.

Methods: An experimental study was conducted at Ethiopian Public Health Institute (EPHI) from September 25 to October 15, 2020. A total of 188 Oro-pharyngeal swabs were collected from COVID-19 suspected cases, referred to EPHI for SARS COV-2 testing. One batch of the sample was inactivated at 56 °C heat for 30 min, and the other batch was stored at 4 °C for a similar period of time. RNA extraction and detection were done by DAAN Gene kit protocols. Abbott m2000 RT-PCR was used for amplification and detection. Data analysis was done by using SPSS version 23.0; Chi-square and Pearson correlation test for qualitative and semi-quantitative analysis were used. p-value < 0.05 was considered as statistically significant.

Results: Out of 188 total samples, 119 (63.3%) were positive and 69 (36.7%) were negative in the non-inactivated group. While, 115 (61.2%) of samples were positive and 73 (38.8) were negative in heat inactivated sample batch. Rate of positivity between groups did not have statistically significant difference (p > 0.05). The mean Cycle threshold (Ct) value difference between the two groups of ORF1a/b gene and N gene was 0.042 (95% CI - 0.247-0.331; t = 0.28; p = 0.774) and 0.38 (95% CI 0.097-0.682; t = 2.638; p = 0.010) respectively.

Conclusion: Heat inactivation at 56 °C for 30 min did not affect the qualitative rRT-PCR detection of SARS-CoV-2. However, the finding showed that there was statistically significant Ct value increment after heat inactivation compared to untreated samples. Therefore, false-negative results for high Ct value (Ct > 35) samples were found to be the challenge of this protocol. Hence alternative inactivation methods should be investigated and further studies should be considered.

Keywords: COVID-19; Ct value; Heat inactivation; SARS-CoV-2; rRT-PCR.

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Conflict of interest statement

All authors declare that they have no conflict or competing interests.

Figures

Fig. 1
Fig. 1
Bland Altman plot of Ct value comparisons between heat-inactivated at 56 °C for 30 min and non-inactivated group in COVID-19 testing, Ethiopia, 2020. A) ORF1a/b gene Ct value comparisons between heat treated group and non-inactivated group. B) N gene Ct value comparisons between heat treated group and non-inactivated group
Fig. 2
Fig. 2
Correlation of inactivated and non-inactivated ORF1a/b gene Ct for Oro-pharyngeal specimen in COVID-19 testing, Ethiopia, 2020
Fig. 3
Fig. 3
Correlation of inactivated and non-inactivated N gene Ct for Oro-pharyngeal specimen in COVID-19 testing, Ethiopia, 2020
Fig. 4
Fig. 4
Correlation of inactivated and non-inactivated ORF1a/b gene Ct value greater than 30 for Oro-pharyngeal specimen in COVID-19 testing, Ethiopia, 2020
Fig. 5
Fig. 5
Correlation of inactivated and non-inactivated N gene Ct value greater than 30 for Oro-pharyngeal specimen in COVID-19 testing, Ethiopia, 2020
See this image and copyright information in PMC

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