Skip to main content

Effect of heat inactivation and bulk lysis on real-time reverse transcription PCR detection of the SARS-COV-2: an experimental study

Abstract

Objective

This study aimed to investigate the effect of heat inactivation and chemical bulklysis on SARS-CoV-2 detection.

Results

About 6.2% (5/80) of samples were changed to negative results in heat inactivation at 60 °C and about 8.7% (7/80) of samples were changed to negative in heat inactivation at 100 °C. The Ct values of heat-inactivated samples (at 60 °C, at 100 °C, and bulk lysis) were significantly different from the temperature at 56 °C. The effect of heat on Ct value should be considered when interpreting diagnostic PCR results from clinical samples which could have an initial low virus concentration. The efficacy of heat-inactivation varies greatly depending on temperature and duration. Local validation of heat-inactivation and its effects is therefore essential for molecular testing.

Introduction

Background

Coronavirus disease 2019 (COVID-19) is a newly emerged human infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (1). SARS-COV-2 is a single-stranded Ribonucleic acid (ssRNA). Based on the rapid rate of increase in humans, the World Health Organization (WHO) classified the COVID-19 outbreak as a pandemic by the end of 2019 [1,2,3]. Since it reached a critical point in March 2020, WHO declared that the world needs speedy and quick solutions to diagnose and tackle the further spread of COVID-19 [4, 5]. The nucleocapsid region and the open reading frame (ORF)-1 of SARS-COV-2 are the most ideal amplification target [6, 7].

The CDC rRT-PCR panel for detection of SARS-CoV-2 demonstrated high sensitivity and specificity for detecting RNA with no observed false-positive reaction [8]. These assays have proven to be valuable for rapid laboratory diagnosis and control of COVID-19 [9, 10]. Laboratory viral nucleic acid (NA) testing using RT-PCR assays is currently the “gold standard” for the diagnosis of COVID-19 [1, 4, 11, 12].

Buffer-based NA extraction methods to obtain high-quality NA has not been developed primarily for the inactivation of infectious samples [13, 14]. Automated NA extraction is often performed outside of the biosafety cabinet. To avoid aerosol formation, a pre-inactivation step under appropriate biosafety conditions is an absolute requirement [13,14,15,16].

Accordingly, the extraction of viral RNA requires the first step of lysis or heat inactivation of the virus at different temperatures and minutes [7, 17]. In our laboratory at the Ethiopian Public Health Institute (EPHI), we usually do heat inactivation for 30 min at 56 °C. The impact of a higher temperature on SARS-COV-2 detection has not been thoroughly examined [17, 18]. Still, there is a lack of understanding of the molecular-level changes that are taking place in the virus due to the different heat and chemical conditions [19, 20].

The effect of heating at different temperatures and time periods prior to testing remains unclear (still under investigation). Thus, this study aims to evaluate the effect of heat inactivation at different temperatures and times and to determine the effect of chemical inactivation by bulk lysis on SARS-CoV-2 detection.

Main text

Materials and methods

Study area and design

The laboratory-based experimental study design was conducted at EPHI National HIV Reference laboratory from August to November 2020. The laboratory has a well-established quality system and is ISO 15189; 2012 accredited for HIV Viral load testing and early infant diagnosis.

Sample size determination and sampling method

Eighty Nasopharyngeal/Oropharyngeal swab samples were selected and taken out from -800C storage. Positive samples with known threshold cycle (CT) value were selected by simple random sampling technique, from 9,520 positive samples within one month, from 28.4% prevalence of Covid-19 in Ethiopia as of August 20/2020. About 2380 samples were done in one week, then dividing this sample by 80 is 30, which was the interval number by which samples were selected. The first sample was selected by the lottery method.

Specimen collection and testing

All samples were tested with 2 controls (1 positive, 1 negative) when testing by reference method (Abbott Real-Time SARS-COV-2(EUA)). Figure 1 Depicts the overall experimental procedure followed.

Fig. 1
figure 1

Process of specimen testing

Fig. 2
figure 2

Mean cyclic threshold comparison between the three temperatures and bulklysis. (The standard errors bar shows mean ± SD): T1-Temperature1 = at 56 °C; T2-temperature 2 = at 60 °C and T3- temperature 3 = at 100 °C; SD standard deviation)

Experiment

Tests were performed using the Abbott Real-time SARS-CoV-2 assay, a rRT-PCR test for the qualitative detection of SARS-CoV-2 in the samples [21]. A dual target assay for RNA-dependent RNA polymerase (RdRp) and N-genes detection of NAs from SARS-CoV-2 was analyzed. Results were reported as positive if the Ct value was < 32, and defined as negative if the Ct value was 32 or more, based upon the manufacturer [1].

The effects of heat treatment at different temperatures and durations and chemical inactivation on the SARS-CoV-2 rRT-PCR Ct-value were evaluated. The samples were inactivated at different temperatures and minutes in a water bath (at 56 °C for 30 min (n = 80), at 60 °C for 30 min (n = 80), and at 100 °C for 10 min (n = 80)) and by chemical bulklysis (n = 80). WHO recommends heat inactivation at 56 °C for 30 min and Abbott real-time RT-PCR as the golden method for SARS-COV-2 detection [22,23,24].

After viral heat inactivation, NA extraction was done from a 0.6 ml sample volume on the Abbott m2000SP instrument by using the Abbott mSample Preparation System. On the other hand, Chemical inactivation was performed to see the effect of bulk lysis on the SARS-CoV-2 rRT-PCR. For chemical inactivation, bulk lysis was tested with the appropriate composition of sample and lysis proportion. Each sample was incubated with the bulk lysis buffer at room temperature for 30 min; then, the sample was extracted.

After the extraction was completed, the samples of heat and chemical treated group was detected by Abbott m2000rt (12). The viral RNA was extracted from 500 μL of each sample, and the final elute was 200 μL by using elution buffer [1, 22]. The SARS-CoV-2 and IC-specific probes were each labeled with a different fluorophore (FAM™ (Carboxyfluorescein), ROX™, (Carboxy-X-rhodamine), and VIC® P (Proprietary dye) for target and IC detection [1].

Quality assurance

All laboratory procedures were performed as per the documented SOP as manufacturing recommendations.

Data analysis

Data was analyzed and described by mean and standard deviation (mean ± SD). Normality was assessed for the three temperature scenarios and bulk lysis, and they were violated normality. However, after we transformed by natural logarithm all of them were normally distributed. Repeated measurement analysis of variance was used to assess the mean difference between temperatures and bulk lysis. To identify the place of significant difference post hoc analysis with modified Bonferroni correction was used.

Results

Reverse transcriptase real-time polymerase chain reaction results of heat-inactivated samples at different temperatures and durations

The effect of heat treatment at different temperatures and durations on the SARS-CoV-2 rRT-PCR Ct-value was evaluated. All heat-inactivated samples at 56 °C for 30 min were tested positive. The Ct values of RdRp were 4.37–31.03 CN (cyclic number) at 56 °C for 30 min, 3.68–30.64 CN at 60 °C for 30 min, 3.37–28.74 CN at 100 °C for 10 min. Chemical bulklysis inactivated samples were from 3.62 to 27.74 CN except for those with weak positive samples (Table 1) which were turned to negative results, as compared to the heat-inactivated samples at 56 °C. Heat inactivation methods resulted in the reduction of positive SARS-CoV-2 samples to undetectable levels, especially in weak positive samples. About 6.2% (5/80) and 8.7% (7/80) of samples were changed to negative results in heat inactivation at 60 °C (30 min) and 100 °C (10 min) respectively. A comparison of heat inactivation of weakly positive samples at 56 °C with (at 60 °C, 100 °C, and chemical bulklysis) was summarized (Table 1).

Table 1 Cycle threshold values for RdRp measured from swab samples following heat treatments at different temperatures and chemical bulklysis

The Ct values of heat-inactivated samples at (60 °C, 100 °C) were significantly different from the temperature at 56 °C (as compared to 56 °C) (p = 0.01, p = 0.001) respectively. The place of significant difference was identified by using post hoc analysis with modified Bonferroni correction (Table 2).

Table 2 Mean difference of group with their confidence interval and p-value following heat inactivation at different temperatures and durations EPHI, Ethiopia

Heat inactivation by chemical bulk lysis

All samples were incubated with chemical bulk lysis buffer, (as it was compared with AVL buffer, AVL serves as the standard for the comparison of the diverse chemical inactivation methods). The decline in the viral RNA quantity was observed in some of the samples treated with chemical bulklysis, especially for those with high Ct values. It was shown that 3.7% (3/80) of analyzed samples were turned to negative. There were significant differences between the Ct values of bulklysis and heat inactivation at 56 °C for the RdRp genes (Repeated measure ANOVA; P = 0.02). Following the comparison of bulk lysis to all other forms of inactivation used in this study, only heat-inactivated at 56 °C was significantly different from bulklysis inactivation (Fig. 2).

Discussion

The SARS-CoV-2 disease has as of late risen and quickly spread in people causing a critical danger to universal wellbeing and the economy.

Respiratory specimens have been used to diagnose SARS-CoV-2 infection by Abbott rRT-PCR, and are regarded as the main detection method. Following the rapid global spread of SARS-CoV-2 and the need for universal testing, more and more individuals are exposed to non-inactivated virus samples. The WHO and United States CDC have released laboratory guidelines to mitigate the risk of exposure during diagnostic and research procedures [25,26,27]. The proceeded to require COVID-19 testing worldwide requires the utilization of straightforward and viable inactivation techniques.

It has appeared that within the SARS-COV-2 swab test, the amount of SARS-COV-2 might be decreased at 60 °C for 30 min and in bulklysis, but still irresistible. As it was heating at a temperature of 100 °C for 10 min was able to inactivate it [12].

However, we have been trying to demonstrate that it is possible to ensure the test integrity by applying heat inactivation under several conditions. RT-PCR Ct values are defined as the number of cycles of amplification required for the accumulated fluorescence (produced by target gene amplification) and are inversely related; low Ct values indicate high viral loads and high Ct values indicate low virus NA concentration in the sample [28]. In this study, the Ct value was essentially influenced by warming at 60 °C and 100 °C for those with weak positive samples. This is in agreement with the studies of Pastorino [12]. The less eminent increase in Ct value observed when the virus was heated to 100 °C, can be ascribed to the shorter warming time, this can be in line with the study by Zou et al. [26]. Lower temperature heat treatment combined with chemical inactivation, short-duration high-temperature heat treatments, or chemical inactivation alone may be more suitable to protect RNA integrity and maximize PCR optimization for the discovery of SARS-COV-2 RNA from low-concentration SARS-COV-2 samples. Our results show significant variation in the effect of heat-treatment inactivation on the SARS-CoV-2 detection. This emphasizes the significance of local approval of inactivation strategies and the need for consistency in inactivation protocols.

Weak positive samples may become false negatives in SARS-CoV-2 RT-PCR detection. Our study also has shown that the heat-inactivated samples at 56 °C were consistent with those in heat-inactivated ones at 60 °C, 100 °C, and chemical bulk lysis for low Ct value results, which agrees with the study done by Pastorino, Rao, and Pan [12, 29, 30].

Warming at 100℃ for 10 min would result in untrue negative, which is steady with that of warming at 92℃ for 15 min, the SARS-CoV-2 RNA in a test was dropped altogether as the study done by Zou et al. and Burton [26, 31]. In reality, considers has been proposed that the test cells should pass on and burst within the occasion of moderately high temperature and long terms, driving to the discharge high number of cell nucleases, and after that, a huge sum of RNA debasement, which may contribute to untrue negative in NA detection. We hypothesize that heating at 100℃ for a long period of time lyse a huge number of cells, and leaves out RNA to RNases enzyme shown within the tests.

Although our study showed that heating at 100 ℃ for 10 min was steady with heat at 56 °C, except for those tests with frail positive, strong positive samples appeared an inclination to diminish in Ct values to a few extents, this is in agreement with the study done by Wang [32]. The RNA conservation may be due to the conservation chemical, which contains guanidine isothiocyanate. This proposed that the nearness of the conservation chemical can viably ensure the keenness of the viral NA, in this manner expanding the extent of recognizable NAs. However, numerous components can impact the effect of the lysis buffer, the amount of virus, nature of the network, contact time, and response temperature, concentration/composition of the lysis buffer used.

Conclusion

We found that the effect of heat-inactivation varies greatly depending on temperature and duration. The impact of chosen inactivation method on the sensitivity of subsequent SARS-CoV-2 detection should be assessed locally. The effect of heat on Ct value should be considered when interpreting diagnostic PCR results.

Limitation

Use of the Abbott Real-time SARS-CoV-2 assay is limited to trained personnel.

Performance has only been established with the specimen types listed in the Intended Use. Other specimen types have not been used with this assay.

Availability of data and materials

The original contributions presented in the study are included in this published article /supplementary material. Further inquiries can be directed to the corresponding author.

Abbreviations

CDC:

Center Disease Control and Prevention

CT:

Cyclic threshold

CN:

Cyclic number

EPHI:

Ethiopian Public Health Institute

EUA:

Emergency Use Authorization

IC:

Internal control

ISO:

International Standard for Organization

SARS-COV-2:

Severe Acute Respiratory Syndrome Coronavirus 2

SOP:

Standard Operating Procedures

RNA:

Ribonucleic acid

WHO:

World Health Organization

RT-PCR:

Real-time polymerase chain reaction

RdRp:

RNA dependent RNA polymerase

VTM:

Viral Transport Media

References

  1. Abbott Molecular. Abbott real-time SARS-COV-2. Abbott real-time SARS-CoV-2 emergency use authorization package 236 Insert, REF 09N77-095, 51-608445/R1. Abbott Molecular Inc., Des Plaines, IL; 2020.

  2. Kratzel A, Todt D, V’kovski P, Steiner S, Gultom M, Thao TT, et al. Inactivation of severe acute respiratory syndrome coronavirus-2 by WHO-recommended hand rub formulations and alcohols. Emerg Infect Dis. 2020;26(7):1592–5. https://doi.org/10.3201/eid2607.200915.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  3. Udugama B, Kadhiresan P, Kozlowski HN, Malekjahani A, Osborne M, Li VYC, et al. Diagnosing COVID-19: the disease and tools for detection. ACS Nano. 2020. https://doi.org/10.1021/acsnano.0c02624.

    Article  PubMed Central  PubMed  Google Scholar 

  4. Gates B. Responding to covid-19—a once-in-a-century pandemic? N Engl J Med. 2020;323(16):2019–20.

    Google Scholar 

  5. World meter. Countries where Coronavirus has spread. World meter; 2020.

  6. Case JB, Bailey AL, Kim AS, Chen RE, Diamond MS. Growth, detection, quantification, and inactivation of SARS-CoV-2. J Virol. 2020. https://doi.org/10.1016/j.virol.2020.05.015.

    Article  Google Scholar 

  7. Lalmuanawma S, Hussain J, Chhakchhuak L. Applications of machine learning and artificial intelligences for Covid-19 (SARS-CoV-2) pandemic. Chaos Solitons Fractals. 2020. https://doi.org/10.1016/j.chaos.2020.110059.

    Article  PubMed Central  PubMed  Google Scholar 

  8. World Health Organization. Laboratory testing strategy recommendations for COVID-19. Evaluation of chemical protocols for inactivating SARS-CoV-2 infectious samples. 2020: 6.

  9. Ngo KA, Jones SA, Church TM. Unreliable inactivation of viruses by commonly used lysis buffers. Appl Biosaf. 2017;22(2):56–9.

    Article  Google Scholar 

  10. World Health Organization. Risk-based classification of diagnostics for WHO prequalification of diagnostics. 2014: 1–18.

  11. D’Cruz RJ, Currier AW, Sampson VB. Laboratory testing methods for novel severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2). Cell Dev Biol. 2020. https://doi.org/10.3389/fcell.2020.004686.

    Article  Google Scholar 

  12. Pastorino B, Touret F, Gilles M, Luciani L, Lamballerie X, Charrel RN. Evaluation of chemical protocols for inactivating SARS-COV-2 infectious samples. Viruses. 2020;12(6):624. https://doi.org/10.3390/v12060624.

    CAS  Article  PubMed Central  Google Scholar 

  13. Kumar M, Mazur S, Ork BL, Postnikova E, Hensley LE, Jahrling PB, et al. Inactivation and safety testing of Middle East Respiratory Syndrome Corona virus. J Virol Methods. 2015;223:13–8.

    CAS  Article  PubMed  Google Scholar 

  14. Seo G, Lee G, Kim MJ, Baek S, Choi M, Ku KB, et al. Rapid detection of COVID-19 causative virus (SARS-CoV-2) in human nasopharyngeal swab specimens. ACS Nano. 2020. https://doi.org/10.1021/acsnano.0c02823.

    Article  PubMed Central  PubMed  Google Scholar 

  15. Africa CDC. Africa Joint Continental Strategy for COVID-19 outbreak.

  16. WHO-COVID-19-lab-testing-2020. (Internet). sur: https://apps.who.int/iris/bitstream/handle/10665/331509/WHO-COVID-19-lab-testing-2020.1-eng.pdf.

  17. Chan KH, MalikPeiris JS, Lam SY, Poon LLM, Yuen KY, Seto WH. The effects of temperature and relative humidity on the viability of the SARS Coronavirus. Adv Virol. 2011. https://doi.org/10.1155/2011/734690.

    Article  PubMed Central  PubMed  Google Scholar 

  18. Kampf G, Voss A, Scheithauer S. Inactivation of corona virus by heat. J Hosp Infect. 2020;105(2):348–9.

    CAS  Article  PubMed  Google Scholar 

  19. Rath SL, Kumar K. Investigation of the effect of temperature on the structure of SARS-CoV-2 spike protein by molecular dynamics simulations. Front Mol Biosci. 2020. https://doi.org/10.3389/fmolb.2020.583523.

    Article  PubMed Central  PubMed  Google Scholar 

  20. Chan KH, Sridhar S, Zhang RR, Chu H, Fung AY-F, Chan G, et al. Factors affecting stability and infectivity of SARS-CoV-2. J Hosp Infect. 2020. https://doi.org/10.1016/j.jhin.2020.07.009.

    Article  PubMed Central  PubMed  Google Scholar 

  21. Umaru FA. Scaling up testing for COVID-19 in Africa: responding to the pandemic in ways that strengthen health systems. Afr J Lab Med. 2020;9(1):19–20.

    Article  Google Scholar 

  22. Chen H, Wu R, Xing Y, Du Q, Xue Z, Xi Y, et al. Influence of different inactivation methods on severe acute respiratory syndrome Corona virus 2 RNA copy number. J Clin Microbiol. 2020. https://doi.org/10.1128/JCM.00958-20.

    Article  PubMed Central  PubMed  Google Scholar 

  23. Hu X, An T, Situ B, Hu Y, Ou Z, Li Q, et al. Heat inactivation of serum interferes with the immunoanalysis of antibodies to SARS-CoV-2. J Clin Lab Anal. 2020. https://doi.org/10.1101/2020.03.12.20034231.

    Article  PubMed Central  PubMed  Google Scholar 

  24. Lu X, Wang L, Sakthivel SK, Whitaker B, Murray J, Kamili S, et al. Real-time reverse transcription PCR panel for detection of severe acute respiratory syndrome Corona virus 2. 2020;26(8). doi: https://doi.org/10.1101/2020.05.19.20101469.

  25. PG. S. Interim laboratory biosafety guidelines for handling and processing specimens associated with Corona virus Disease 2019 (COVID-19).

  26. Zou J, Zhi S, Chen M, Su X, Kang L, Li C, et al. Heat inactivation decreases the qualitative real-time RT-PCR detection rates of clinical samples with high cycle threshold values in COVID-19. Diagn Microbiol Infect Dis. 2020;98(1):115109.

    CAS  Article  PubMed  Google Scholar 

  27. World Health Organization. Laboratory testing for coronavirus disease 2019 (COVID-19) in suspected human cases: interim guidance. 2020.

  28. United States Centers for Disease Control and Prevention. Interim laboratory biosafety guidelines for handling and processing specimens associated with coronavirus disease 2019 (COVID-19). 2020.

  29. Rao SN, Manissero D, Steele VR, Pareja J. A systematic review of the clinical utility of cycle threshold values in the context of COVID-19. Infect Dis Ther. 2020;9:573–86.

    Article  PubMed  Google Scholar 

  30. Pan Y, Long L, Zhang D, Yan T, Cui S, Yang P, et al. Potential false-negative nucleic acid testing results for severe scute respiratory syndrome coronavirus 2 from thermal inactivation of samples with low viral loads. J Clin Chem. 2020;66(6):794–801. https://doi.org/10.1093/clinchem/hvaa091.

    Article  Google Scholar 

  31. Burton J, Love H, Richards K, Burton C, Summers S, Pitman J, et al. The effect of heat-treatments on SARS-CoV-2 viability and detection. J Virol Methods. 2021;290: 114087. https://doi.org/10.1016/j.jviromet.2021.114087.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  32. Wang T, Lien C, Liu S, Selvaraj P. Effective heat inactivation of SARS-CoV-2. medRxiv. 2020. https://doi.org/10.1101/2020.04.29.20085498.

Download references

Acknowledgements

We are grateful in extending our thanks to Ethiopian Public Health Institute for allowing as the setup, ethically approve our research and allow as to use the leftover samples. We also extend our appreciation to Mr. Atsbeha G/eigziebxier for his unreserved contribution.

Funding

The author(s) received no specific funding for this work.

Author information

Authors and Affiliations

Authors

Contributions

DL has participated in the design, coordination of the study, and data collection, analysis, and interpretation. GG has contributed in, drafting, revising, or critically reviewing the article and revision and amendment of the draft manuscript. GGH has contributed in wright up, analysis, and review, RD has contributed in wright up, analysis, and review, GB has contributed in data collection and analysis, TS has contributed in data collection and analysis, DA has contributed in data collection and analysis, DC has contributed in data collection, analysis, and manuscript preparation, BL has contributed in wright up, analysis and review, JB has contributed to conceptualization and methodology, SA has contributed to conceptualization and writeup, HHT has contributed to the design, including final approval of the version to be published. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Dereje Leta.

Ethics declarations

Ethics approval and consent to participate

Ethical clearance was obtained from EPHI Scientific ethical review committee. Approval and official permission was obtained from the EPHI research committee to use the leftover samples in the laboratory (EPHI-IRB-296–2020). The confidentiality of the data collected was kept to a maximum and each patient identity was coded.

Facilities available for the study

The study was conducted in the Ethiopian public health institute. We were also, using suitable data storage devices and a recent version of statistical packages that are available at EPHI for data storage and analysis.

Consent to publish

Not applicable.

Competing interests

"The authors declare that they have no competing interests".

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Leta, D., Gutema, G., Hagos, G.G. et al. Effect of heat inactivation and bulk lysis on real-time reverse transcription PCR detection of the SARS-COV-2: an experimental study. BMC Res Notes 15, 295 (2022). https://doi.org/10.1186/s13104-022-06184-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13104-022-06184-z

Keywords

  • Heat inactivation
  • SARS-COV-2
  • RT- PCR
  • Ct value