Reliability of RT-qPCR Pooling Method for COVID-19 Detection in Various Cycle Threshold Values
Background: Reverse Transcriptase Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) is a standard method to detect SARS-CoV-2, the cause of COVID-19 disease, albeit expensive for some laboratory settings. The pooling test is widely used for large-scale screening to speed up the turn-around...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Universitas Padjadjaran
2023-06-01
|
| Series: | Althea Medical Journal |
| Subjects: | |
| Online Access: | https://journal.fk.unpad.ac.id/index.php/amj/article/view/2940 |
| _version_ | 1797372703983796224 |
|---|---|
| author | Muhammad Fauzan Alif Radjawali Muti'ah Jihadah Lidya Chaidir |
| author_facet | Muhammad Fauzan Alif Radjawali Muti'ah Jihadah Lidya Chaidir |
| author_sort | Muhammad Fauzan Alif Radjawali |
| collection | DOAJ |
| description | Background: Reverse Transcriptase Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) is a standard method to detect SARS-CoV-2, the cause of COVID-19 disease, albeit expensive for some laboratory settings. The pooling test is widely used for large-scale screening to speed up the turn-around time and reduce the cost of the RT-qPCR. However, the pooling test involves mixing a certain number of specimens which theoretically increases the possibility of false-negative results. This study aimed to evaluate the accuracy of the pooling test compared with the non-pooling test in different Ct values as a surrogate for viral load.
Methods: RT-qPCR was performed in three groups of samples: non-pooling (individual samples), pooling of 5 samples and 11 samples, with various ranges of Ct value in the respective group: x<25 (n=4); 25<x<30 (n=5), x<30 (n=16), and negative sample (n=5). Agreement and kappa values were calculated. Four of twenty-five individual samples resulted in false-negative after pooling.
Results: By taking all samples without applying the cut-off value to the calculation, the agreement in pooling of 5 samples was 0.86 (Kappa 0.31) and of 11 samples was 0.64 (Kappa 0.96). When the cut-off value of Ct<37 was applied, percent agreement and kappa were 1.00, respectively, for both pooling methods.
Conclusions: Pooling up to 11 samples shows high concordance with RT-qPCR with individual samples with Ct<37. Interpreting pooling results in a very low viral load (Ct≥37) must be considered due to the increased possibility of inconclusive results. |
| first_indexed | 2024-03-08T18:39:39Z |
| format | Article |
| id | doaj.art-20f4d818132544019c1020d9f501f7c6 |
| institution | Directory Open Access Journal |
| issn | 2337-4330 |
| language | English |
| last_indexed | 2024-03-08T18:39:39Z |
| publishDate | 2023-06-01 |
| publisher | Universitas Padjadjaran |
| record_format | Article |
| series | Althea Medical Journal |
| spelling | doaj.art-20f4d818132544019c1020d9f501f7c62023-12-29T08:54:27ZengUniversitas PadjadjaranAlthea Medical Journal2337-43302023-06-0110210.15850/amj.v10n2.29401595Reliability of RT-qPCR Pooling Method for COVID-19 Detection in Various Cycle Threshold ValuesMuhammad Fauzan Alif Radjawali0Muti'ah Jihadah1Lidya Chaidir2Faculty of Medicine, Universitas PadjadjaranCenter for Translational Biomarker Research, Universitas PadjadjaranDepartment of Biomedical Sciences, Faculty of Medicine Universitas PadjadjaranBackground: Reverse Transcriptase Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) is a standard method to detect SARS-CoV-2, the cause of COVID-19 disease, albeit expensive for some laboratory settings. The pooling test is widely used for large-scale screening to speed up the turn-around time and reduce the cost of the RT-qPCR. However, the pooling test involves mixing a certain number of specimens which theoretically increases the possibility of false-negative results. This study aimed to evaluate the accuracy of the pooling test compared with the non-pooling test in different Ct values as a surrogate for viral load. Methods: RT-qPCR was performed in three groups of samples: non-pooling (individual samples), pooling of 5 samples and 11 samples, with various ranges of Ct value in the respective group: x<25 (n=4); 25<x<30 (n=5), x<30 (n=16), and negative sample (n=5). Agreement and kappa values were calculated. Four of twenty-five individual samples resulted in false-negative after pooling. Results: By taking all samples without applying the cut-off value to the calculation, the agreement in pooling of 5 samples was 0.86 (Kappa 0.31) and of 11 samples was 0.64 (Kappa 0.96). When the cut-off value of Ct<37 was applied, percent agreement and kappa were 1.00, respectively, for both pooling methods. Conclusions: Pooling up to 11 samples shows high concordance with RT-qPCR with individual samples with Ct<37. Interpreting pooling results in a very low viral load (Ct≥37) must be considered due to the increased possibility of inconclusive results.https://journal.fk.unpad.ac.id/index.php/amj/article/view/2940cycle threshold (ct) value, covid-19, pooling test, rt-qpcr |
| spellingShingle | Muhammad Fauzan Alif Radjawali Muti'ah Jihadah Lidya Chaidir Reliability of RT-qPCR Pooling Method for COVID-19 Detection in Various Cycle Threshold Values Althea Medical Journal cycle threshold (ct) value, covid-19, pooling test, rt-qpcr |
| title | Reliability of RT-qPCR Pooling Method for COVID-19 Detection in Various Cycle Threshold Values |
| title_full | Reliability of RT-qPCR Pooling Method for COVID-19 Detection in Various Cycle Threshold Values |
| title_fullStr | Reliability of RT-qPCR Pooling Method for COVID-19 Detection in Various Cycle Threshold Values |
| title_full_unstemmed | Reliability of RT-qPCR Pooling Method for COVID-19 Detection in Various Cycle Threshold Values |
| title_short | Reliability of RT-qPCR Pooling Method for COVID-19 Detection in Various Cycle Threshold Values |
| title_sort | reliability of rt qpcr pooling method for covid 19 detection in various cycle threshold values |
| topic | cycle threshold (ct) value, covid-19, pooling test, rt-qpcr |
| url | https://journal.fk.unpad.ac.id/index.php/amj/article/view/2940 |
| work_keys_str_mv | AT muhammadfauzanalifradjawali reliabilityofrtqpcrpoolingmethodforcovid19detectioninvariouscyclethresholdvalues AT mutiahjihadah reliabilityofrtqpcrpoolingmethodforcovid19detectioninvariouscyclethresholdvalues AT lidyachaidir reliabilityofrtqpcrpoolingmethodforcovid19detectioninvariouscyclethresholdvalues |