SARS-CoV-2 virus in Finnish wastewater: Optimization of RT-ddPCR method and comparison with the RT-qPCR method in virus detection
Janhonen, Erja (2021)
2021
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2021-05-04
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202104273879
https://urn.fi/URN:NBN:fi:tuni-202104273879
Tiivistelmä
Background and aims: SARS-CoV-2 is a single-stranded envelope RNA virus that causes currently occurring COVID-19 pandemic. Those infected with the virus excrete it in wastewater through urine, feces, saliva and various body secretions, and thus the virus is transmitted to the sewer system. Asymptomatic can also secrete the virus. At the population level, the presence of the virus and its concentration in wastewater can be studied from the samples collected from the wastewater treatment plants. The advantage of wastewater monitoring versus clinical testing take in healthcare is greater coverage, and also it detects patients who have not applied for the test. The aim of this work was to study the detection of the virus by the droplet digital PCR (ddPCR) and to compare the method to the more commonly used quantitative PCR (qPCR) method. The results of these methods were compared to the regional COVID-19 statistics of the Infectious Disease Registry of Finland.
Materials and methods: In the method optimization part of the work the most sensitive way to detection of SARS-CoV-2 RNA in the wastewater samples by ddPCR is searched. SARS-CoV-2 RNA isolated from patient samples as well as wastewater samples were used for the method optimization and detection of PCR inhibition. The optimized methods were compared to the methods used in the international laboratories using QCMD test panel. The thesis work compares the differences between PCR primers recognizing different gene regions of SARS-CoV-2. Wastewater samples from the wastewater treatment plants were concentrated by a Centricon Plus -70 filter and RNA was isolated from the samples using the Qiagen Viral Mini RNA Kit and an isolation robot with Chemagic Viral RNA/DNA Kit. The presence of SARS-CoV-2 in the samples and its concentrations were studied by ddPCR and qPCR, and sensitivity and specificity of these methods were compared. The epidemiological part of the work compares the obtained results with the regional COVID-19 situation.
The results: N1 and E PCR primers showed the best performance in ddPCR, of which N1 was slightly more sensitive. As a virus isolation method in ddPCR, the Qiagen Viral Kit performed somewhat better than the Chemagic Viral Kit. N2 has been the most functional PCR primer in qPCR, although it proved to be insensitive in ddPCR. In qPCR slightly better accuracy was obtained using the Chemagic Viral Kit than the Qiagen Viral Kit. In the test performed, no inhibition of wastewater was observed with ddPCR. ddPCR detected low viral concentrations, 1:80 000 diluted RNA at the lowest and it was as sensitive as the detection limit values given in the literature. The lowest detected amount of viral RNA was 1:5 000 clinical sample by qPCR so ddPCR was more sensitive than qPCR. Both methods proved to be specific in detecting all SARS-CoV-2 positive samples in the test and both gave negative results to samples containing other coronaviruses. SARS-CoV-2 was present in 16/20 of the samples which had been collected on February and March 2021 from the wastewater treatment plants (WWTPs). These WWTPs were Oulu, Helsinki, Espoo, Tampere, Turku, Kuopio, Lappeenranta and Rovaniemi. According to the Infectious Disease Registry of Finland, COVID-19 cases occurred in the areas of these treatment plants. No virus was detected in Seinäjoki and Pietarsaari and according to the Infectious Disease Registry, there were no cases in Pietarsaari in either week or in Seinäjoki in March. In Seinäjoki there were 7 cases in February and result of the sample was negative.
Conclusion: ddPCR is a sensitive and a specific method in detection of SARS-CoV-2. The highest accuracy on this work was obtained using N1 primer and the Qiagen Viral Kit. The method detects the virus and its concentration in the wastewater as well an in the clinical samples. In addition to sensitivity and specificity, the method has the advantage of good tolerance to PCR inhibitors. The challenges of the method compared to qPCR are multi-step, higher cost, and longer analysis time. The method detected the virus in 8 WWTPs. The prevalence of SARS-CoV-2 in wastewater correlates with diagnosed COVID-19 cases and virus monitoring is a promising method to monitor the development of a population-level SARS-CoV-2 pandemic.
Materials and methods: In the method optimization part of the work the most sensitive way to detection of SARS-CoV-2 RNA in the wastewater samples by ddPCR is searched. SARS-CoV-2 RNA isolated from patient samples as well as wastewater samples were used for the method optimization and detection of PCR inhibition. The optimized methods were compared to the methods used in the international laboratories using QCMD test panel. The thesis work compares the differences between PCR primers recognizing different gene regions of SARS-CoV-2. Wastewater samples from the wastewater treatment plants were concentrated by a Centricon Plus -70 filter and RNA was isolated from the samples using the Qiagen Viral Mini RNA Kit and an isolation robot with Chemagic Viral RNA/DNA Kit. The presence of SARS-CoV-2 in the samples and its concentrations were studied by ddPCR and qPCR, and sensitivity and specificity of these methods were compared. The epidemiological part of the work compares the obtained results with the regional COVID-19 situation.
The results: N1 and E PCR primers showed the best performance in ddPCR, of which N1 was slightly more sensitive. As a virus isolation method in ddPCR, the Qiagen Viral Kit performed somewhat better than the Chemagic Viral Kit. N2 has been the most functional PCR primer in qPCR, although it proved to be insensitive in ddPCR. In qPCR slightly better accuracy was obtained using the Chemagic Viral Kit than the Qiagen Viral Kit. In the test performed, no inhibition of wastewater was observed with ddPCR. ddPCR detected low viral concentrations, 1:80 000 diluted RNA at the lowest and it was as sensitive as the detection limit values given in the literature. The lowest detected amount of viral RNA was 1:5 000 clinical sample by qPCR so ddPCR was more sensitive than qPCR. Both methods proved to be specific in detecting all SARS-CoV-2 positive samples in the test and both gave negative results to samples containing other coronaviruses. SARS-CoV-2 was present in 16/20 of the samples which had been collected on February and March 2021 from the wastewater treatment plants (WWTPs). These WWTPs were Oulu, Helsinki, Espoo, Tampere, Turku, Kuopio, Lappeenranta and Rovaniemi. According to the Infectious Disease Registry of Finland, COVID-19 cases occurred in the areas of these treatment plants. No virus was detected in Seinäjoki and Pietarsaari and according to the Infectious Disease Registry, there were no cases in Pietarsaari in either week or in Seinäjoki in March. In Seinäjoki there were 7 cases in February and result of the sample was negative.
Conclusion: ddPCR is a sensitive and a specific method in detection of SARS-CoV-2. The highest accuracy on this work was obtained using N1 primer and the Qiagen Viral Kit. The method detects the virus and its concentration in the wastewater as well an in the clinical samples. In addition to sensitivity and specificity, the method has the advantage of good tolerance to PCR inhibitors. The challenges of the method compared to qPCR are multi-step, higher cost, and longer analysis time. The method detected the virus in 8 WWTPs. The prevalence of SARS-CoV-2 in wastewater correlates with diagnosed COVID-19 cases and virus monitoring is a promising method to monitor the development of a population-level SARS-CoV-2 pandemic.