Dhibika M, Madhusudhan N S, Malini A, Natarajan Mailan
Microbiology, Indira Gandhi Medical College & Research Institute, Puducherry, IND.
Cureus. 2023 Mar 27;15(3):e36773. doi: 10.7759/cureus.36773. eCollection 2023 Mar.
Objectives 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 methods A 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 analysis Data 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. Results The 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. Conclusion The 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 cross-contamination and technical errors.
目的 在新冠疫情期间，多个实验室根据现有资源采用了不同的RNA提取方法。因此开展本研究以比较手动和自动方法在qRT-PCR中的Ct值、所需时间（样本处理至PCR上样）、人力需求以及耗材成本。
材料与方法 对VRDL接收的120份用于RT-PCR检测的鼻咽/口咽拭子进行横断面研究。基于自动RNA提取（Genetix，HT 96纯化仪）和RT-PCR（Trivitron PCR试剂盒）检测E基因（筛查）和ORF基因（确证）的结果，分为I组（Ct 15 - 22）、II组（Ct 23 - 29）、III组（Ct 30 - 36）和IV组（Ct >36）。使用磁珠（Lab system，Trivitron）进行手动RNA提取。
统计分析 采用SPSS 19.0版软件进行数据分析。通过配对t检验对两种方法获得的Ct值进行组间比较。采用Z检验比较其他参数。
结果 I组至III组中，目标基因Ct值的差异具有统计学意义（p<0.05）；然而，结果解读无差异。时间、人力和成本的差异具有统计学意义（p<0.05）。手动方法所需人力多一倍；时间多40分钟，自动方法的耗材成本高3.5倍。
结论 研究表明，与手动提取相比，自动提取的RNA产量更高。通过自动方法提取的样本在PCR检测中比手动方法能在更低的Ct范围内检测到病毒。自动方法处理样本所需时间更少，人力需求也更少。考虑到成本因素，在资源有限的环境中手动提取可能更可取，因为检测结果没有差异。手动方法需要更多的实际操作时间，存在交叉污染和技术错误的潜在风险。
