key: cord-0765816-9q1qgl9y authors: Dong, Wenxue; Yang, Xu; Li, Jing; Zhang, Zhiying; Liu, Lijun; Zhao, Zhipeng; Kang, Longli title: Smaller reaction volume of triplex taqman real‐time reverse transcription‐PCR assays for diagnosing coronavirus disease 2019 date: 2021-12-03 journal: J Clin Lab Anal DOI: 10.1002/jcla.24137 sha: da176a5c0703d18d6dea22db50927b228f4b8516 doc_id: 765816 cord_uid: 9q1qgl9y BACKGROUND: Coronavirus disease 2019 (COVID‐19) has had a devastating impact on public health services worldwide. Currently, there are no standard remedies or therapies for COVID‐19. it is important to identify and diagnose COVID‐19 to control the spread. But clinical symptoms of COVID‐19 are very similar to those of other respiratory viruses. RESULTS: As a result, the diagnosis of COVID‐19 relies heavily on detecting pathogens. We established a bunch of triplex new TaqMan real‐time PCR assays. Three sets of primers and probes (targeting the ORF1ab, N, and E genes, respectively) are poorly consistent with other human coronaviruses and the human influenza virus. The sensitivity of established PCR assays notices as few as 100 copies per PCR of the ORF1ab, N, and E genes. Meanwhile, standard curves concluded from constant PCR reaction all showed glorious linear correlations between Ct values and the polymer loading copy variety (correlation coefficient (R(2)) of ORF1ab, N, and E genes is 0.996, 0.991, and 0.998, respectively). Surveillance of RNA‐based pseudovirus demonstrated that they were identified to be positive with respect to SARS‐CoV‐2 and that established PCR assays are achievable. CONCLUSION: The assays established provide a smaller reaction volume for diagnosing COVID‐19. proteins (envelope, E; membrane, M; nucleocapsid phosphoprotein, N; spike, S). 4 However, compared to SARS-CoV or MERS-CoV, SARS-CoV-2 spreads sooner making it troublesome to regulate. 5 Several studies have shown that the majority of the COVID-19 patients can present with fever, cough, fatigue, headache, myalgia, and severe respiratory disease. [6] [7] [8] The most common manifestations of COVID-19 patients are almost similar to SARS and MERS. 9 Therefore, the diagnosis of COVID-19 is basically supported by the detection of pathogens. The World Health Organization and, therefore, the Chinese Centers for Disease Control have developed several RT-PCR protocols to diagnose COVID-19, which was helpful in identifying individuals at potential risk for SARS-CoV-2 transmission. 10 However, these applications are also critically affected by the high accuracy of the test. With respect to clinical decision-making, all of the causes of test failure led to erroneous diagnoses that gave rise to false positives or false negatives. We established another set of triplex new TaqMan real-time PCR assays to identify SARS-CoV-2, and to conduct an evaluation of the availability of real-time PCR assays. In this study, the reference sequences, available through the National Center for Biotechnology Information (NCBI; NC_045512.2, SARS-CoV-2), were synthesized (TsingKe Biotech, China) and provided templates for assay design. Target regions ORF1ab, N, and E DNA samples from SARS-CoV-2 have been amplified with the PCR specific primers (forward and reverse). Target regions of ORF1ab, N, and E, synthesized by TsingKe Biotech within the same vector (pcoldII), were subcloned within the plasmid (pET21a) using the EcoR I and Xho I restriction site. It was confirmed by next-generation sequencing (NGS), and subsequently reworked into the E. coli DH5α (Tiangen Biotech). The plasmid was exploited using TIANprep Mini Plasmid Kit (DP103, Tiangen Biotech), quantitated by ultraviolet (UV) light spectroscopy, and diluted by 10-fold gradient dilution for reserve use. The target sequence of ORF1ab, N, and E was synthesized and cloned into a lentiviral vector and pseudovirus was prepared in 293T cells. This is a product called 2019- Three sets of primers and probes (targeting ORF1ab, N, and E gene, respectively), supported the ordination of SARS-CoV-2 (NCBI: NC_045512.2), were designed by NCBI Primer-BLAST (https://www. ncbi.nlm.nih.gov/tools/ prime r-blast/ index.cgi?LINK_LOC=Blast Home). Table 1 provides all primers and probes synthesized by TsingKe Biotech using the phosphoramidite method. All primers and TaqMan probe sequences were confirmed by com- In order to determine the sensitivity of the developed RT-PCR detection, the limit of detection was analyzed by 10-fold dilutions of plasmid from the E, N, and ORF1ab genes as models. Table 2 and Figure 1 demonstrate that the RT-PCR assay was sensitive, detecting as few as 100 copies per PCR of the ORF1ab, N, and E genes. The PCR assays were repeated thrice at 100 copies/reac- In order to ascertain the reproducibility of PCR assays, intra-assay and interassay variabilities were assessed. The PCR assays were repeated thrice and linear regression analysis indicated that 100% repeatability was achieved when the range was greater than 100 copies/reactions for the ORF1ab, N, and E gene assays. The results of the correlational analysis are presented in Table 3 . The coefficient of variation (CV) of Ct values within and between reactions was 0.224% ~1.231% and 1.134% ~6.503%, respectively, for the ORF1ab gene, 0.076% ~11.948% and 0.679% ~11.722%, respectively, for the N gene, and 0.011% ~1.223%, respectively, for the E gene. These suggest that the assays developed are highly reproducible. The specificity of TaqMan primers and probes was confirmed by comparing the gene regions of all known SARS-CoV-2 sequences in the NCBI gene bank. All signed primers and probe sequences are identical to all published sequences of SRAS-CoV-2. We lined up our primers and probes with the reference sequences of other human coronaviruses and flu viruses. Figure 2 shows that the aforementioned viral genome has poor consistency with primers and probes developed during this study, except for the SARS E gene. When the ORF1ab, N, and E genes were all positive in the assays, it was believed to be an infection with SARS-CoV-2. The latter suggests that the assays developed are special. RNA-based pseudovirus has been evaluated through PCR assays. Positive results were confirmed for SARS-CoV-2. It demonstrates that the PCR assays is achievable (Figure 3 ). When RNA was F I G U R E 1 The PCR amplification curve originated from 10-fold dilution of plasmid from the ORF1ab gene assay (A), N gene assay (B), and E gene assay (C) of SARS-CoV-2 extracted from a 10-fold gradient dilution series of the pseudovirus and was reverse-transcribed to the cDNA to determine the detection limit of the PCR assays, the results showed that the detection limit of ORF1ab, N, and E gene is 3.75 × 10 5 , 2.76 × 10 5 , 8.775 × 10 5 copies/reactions, respectively. This is different from the detection limit of DNA plasmid as a template, perhaps because of the low performance of RNA extraction. Reported incidents of COVID-19 continue to escalate, posing a serious public health threat. 11 We developed a triplex TaqMan real-time PCR assay with a smaller volume of reaction mixture. This helps to identify and diagnose COVID-19 quickly, accurately, and on a preferred basis. Two TaqMan real-time RT-PCR assays, developed by Liu et al, have an N gene detection limit of 250 copies/ml. 13 The sensitivity of commercial kits ranges from 10 2 to 10 3 copies/ml. In conclusion, we have developed triplex real-time TaqMan RT-PCR tests that help to identify and diagnose COVID-19 quickly, accurately, and on a preferred basis. The authors have contributed equally to this work. Longli Kang and Zhipeng Zhao were responsible for design. Zhiying Zhang, Jing Li, Junli Liu, Wenxue Dong, and Xu Yang were responsible for implementing and analyzing data. Wenxue Dong wrote the manuscript. Longli Kang provided insight on structuring the manuscript, and all authors contributed to manuscript completion. The datasets supporting the conclusions of this article are included within the article. 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