Comparison of Manual and Automated Nucleic Acid (RNA) Extraction Methods for the Detection of SARS-CoV-2 by qRT-PCR

Abstract

During the COVID-19 pandemic, several laboratories used different RNA extraction methods based on the resources available. Hence this study was done to compare the Ct values in qRT-PCR, time taken (sample processing-loading to PCR), manpower requirement, and cost of consumables between manual and automated methods. Materials and methodsA cross-sectional study was done on 120 nasopharyngeal/oropharyngeal swabs received in VRDL for RT-PCR testing. Based on the results of automated RNA extraction (Genetix, HT 96 Purifier) and RT-PCR (Trivitron PCR Kit) detecting E gene (screening) and ORF gene (confirmatory), the division into Group-I (Ct 15-22), Group-II (Ct 23-29), Group-III (Ct 30-36) and Group-IV (Ct >36) was done. Manual RNA extraction was done using magnetic beads (Lab system, Trivitron). Statistical analysisData were analyzed by SPSS 19.0 version software. Ct values obtained in the two methods were compared by paired t-test, GroupWise. Z test was used to compare the other parameters. ResultsThe difference in Ct values for target genes was statistically significant (p<0.05) in Group-I to III; however, no variation in result interpretation. The difference in time, manpower, and cost were statistically significant (p<0.05). The manual method required twice more manpower; 40 minutes more time & automated method cost 3.5 times more for consumables. ConclusionThe study showed that RNA yield was better with automated extraction in comparison to manual extraction. The samples extracted by the automated method detected the virus at a lower Ct range by PCR than the manual method. Automated method processed samples in less time and with less manpower. Considering the cost factor, manual extraction can be preferred in resource-limited settings as there was no difference in the results of the test. The manual method requires more hands-on time with potential chances of crosscontamination and technical errors.

Evaluation of the time, labor, and money required for manual and automated nucleic acid (RNA) isolation for the detection of SARS-COV-2 by QRT-PCR using the qiamp viral RNA mini kit and kingfisher flex

“…Additionally, the study conducted by Kumar et al, 13 comparing two automated platforms with manual RNA extraction, found comparable efficacy in RNA extraction between the manual method and one of the automated methods. This consistency across studies supports the idea that various RNA extraction methods may yield different Ct values, yet the ultimate interpretation of results remains unaffected.…”

A semi-automated and high-throughput approach for the detection of honey bee viruses in bee samples

“…The magnetic beads-based technology has been widely used in biomedical research [33][34][35][36][37], and has become one of the most used methods to extract viral RNA for screening for SARS-CoV-2 [36], as rapid diagnosis of COVID-19 is essential to restrict its spreading. A same extraction method combined with an automated approach, which is rapid, high-throughput, uniform and bears low cross contamination risk [50][51][52] may be employed for honey bee research, in particular for virus screening surveys to screen multiple colonies in a limited timeframe.…”

Research on a Magnetic Separation-Based Rapid Nucleic Acid Extraction System and Its Detection Applications

“…Automated nucleic acid extraction instruments have the potential to improve workflow and reduce variability in clinical laboratories. Some reports have already identified the value of automated nucleic acid extraction instruments compared to manual methods [23][24][25][26]. Although most reports mention the benefits of streamlining workflows and reducing hands-on time, the quality of extracted nucleic acids can vary.…”