Multicenter post-marketing clinical evaluation on the clinical performance of the five domestic fast nucleic acid detection reagents for 2019-nCoV.

Objective This multicenter clinical evaluation analyzed the clinical performance of five fast nucleic acid detection systems for 2019-nCoV. Methods Clinical performance of the five fast nucleic acid detection reagents approved in China was evaluated in the present study. Fifty-seven throat swabs samples from COVID-19 patients and fifteen throat swabs samples from healthy people were collected from the First Affiliated Hospital of Zhejiang University school of Medicine, Tongji Hospital of Tongji Medical College of HUST, and National Institute of Viral Disease Control and Prevention of CDC to evaluate the positive coincidence rate, negative coincidence rate, total coincidence rate, the detection time and retest rate as well as the relation between positive intensity and positive coincidence rate of the five fast nucleic acid detection systems in November 2020. Results The positive coincidence rates of the five kits were 92.59% (50/54), 83.64% (46/55), 98.25% (56/57), 94.44% (51/54) and 98.18% (54/55);and the negative coincidence rates were 93.33% (14/15), 93.33% (14/15), 86.67% (13/15), 100% (14/14) and 93.33% (14/15);and the total coincidence rates were 92.75% (64/69), 85.71% (60/70), 95.83% (69/72), 94.20% (65/69) and 97.14% (68/70), respectively. The positive coincidence rate of the five kits reached 100% for the strong-positive (90/90) and medium-positive samples (84/84), but only 82.18% (83/101) for weak-positive samples (cycle threshold value>33), and the retest rate of two kits were 15.28% (11/72) and 12.50% (9/72), which were both higher than 10%. Total time from sample extraction to amplification was between 32.33-65.33 minutes for these five kits. Conclusion The five fast nucleic acid detection reagents have good performance and can be used as a supplement to routine nucleic acid detection reagents.Copyright © 2022 Chinese Journal of Laboratory Medicine. All rights reserved.

Development of SYBR green RT-qPCR assay for titrating bivalent live infectious bronchitis vaccines.

Infectious bronchitis (IB) is a highly contagious viral disease of chickens caused by IB virus (IBV) that can cause substantial economic losses in the poultry industry. IBV variant infections have been continuously reported since the initial description in the 1930s. QX-like IBVs are the predominant circulating genotype globally. A homologous QX vaccine has superior protection efficacy compared with that of other available vaccines, and the combination of Massachusetts (Mass)-like and QX-like strains is being used to combat QX-like IBV infections. Inoculation of embryonated chicken eggs is the standard method for the titration of IBV, and the titer is expressed as 50% egg infectious dose (EID50). However, this method cannot effectively distinguish or quantify different genotypic strains in a mixture of different viruses, especially in the absence of neutralizing monoclonal antibodies. In this study, quantitative real-time PCR (RT-qPCR) was applied using specific primers for the QX- and Mass-like strains to quantitate IBV infection and for comparison with the conventional virus titration quantitative method. A strong positive correlation was observed between RT-qPCR cycle threshold values and the different EID50 concentrations. This method was further used to titrate bivalent IB vaccines, and the amount of individual genotype virus was determined based on specific primers. Thus, this RT-qPCR assay may be used as a highly specific, sensitive, and rapid alternative to the EID50 assay for titering IBVs.

Delivery of siRNAs against MERS-CoV in Vero and HEK-293 cells: A comparative evaluation of transfection reagents.

Background: A new coronavirus was identified in Jeddah, Saudi Arabia in 2012 and designated as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). To date, this virus has been reported in 27 countries. The virus transmission to humans has already been reported from camels. Currently, there is no vaccine or antiviral therapy available against this virus. Methods: The siRNAs were in silico predicted, designed, and chemically synthesized by using the MERS-CoV-orf1ab region as a target. The antiviral activity was experimentally evaluated by delivering the siRNAs with Lipofectamine™ 2000 and JetPRIMER as transfection reagents in both Vero cell and HEK-293-T cell lines at two different concentrations (10.0 nM and 5.0 nM). The Ct value of quantitative Real-Time PCR (qRT-PCR) was used to calculate and determine the reduction of viral RNA level in both cell supernatant and cell lysate isolated from both cell lines. Results: The sequence alignment resulted in the selection of highly conserved regions. The orf1ab region was used to predict and design the siRNAs and a total of twenty-one siRNAs were finally selected from four hundred and twenty-six siRNAs generated by online software. Inhibition of viral replication and significant reduction of viral RNA was observed against selected siRNAs in both cell lines at both concentrations. Based on the Ct value, the siRNAs # 11, 12, 18, and 20 were observed to be the best performing in both cell lines at both concentrations. Conclusion: Based on the results and data analysis, it is concluded that the use of two different transfection reagents was significantly effective. But the Lipofectamine™ 2000 was found to be a better transfection reagent than the JetPRIMER for the delivery of siRNAs in both cell lines.

Multicenter post-marketing clinical evaluation on the clinical performance of the five domestic fast nucleic acid detection reagents for 2019-nCoV

Objective This multicenter clinical evaluation analyzed the clinical performance of five fast nucleic acid detection systems for 2019-nCoV. Methods Clinical performance of the five fast nucleic acid detection reagents approved in China was evaluated in the present study. Fifty-seven throat swabs samples from COVID-19 patients and fifteen throat swabs samples from healthy people were collected from the First Affiliated Hospital of Zhejiang University school of Medicine, Tongji Hospital of Tongji Medical College of HUST, and National Institute of Viral Disease Control and Prevention of CDC to evaluate the positive coincidence rate, negative coincidence rate, total coincidence rate, the detection time and retest rate as well as the relation between positive intensity and positive coincidence rate of the five fast nucleic acid detection systems in November 2020. Results The positive coincidence rates of the five kits were 92.59% (50/54), 83.64% (46/55), 98.25% (56/57), 94.44% (51/54) and 98.18% (54/55);and the negative coincidence rates were 93.33% (14/15), 93.33% (14/15), 86.67% (13/15), 100% (14/14) and 93.33% (14/15);and the total coincidence rates were 92.75% (64/69), 85.71% (60/70), 95.83% (69/72), 94.20% (65/69) and 97.14% (68/70), respectively. The positive coincidence rate of the five kits reached 100% for the strong-positive (90/90) and medium-positive samples (84/84), but only 82.18% (83/101) for weak-positive samples (cycle threshold value>33), and the retest rate of two kits were 15.28% (11/72) and 12.50% (9/72), which were both higher than 10%. Total time from sample extraction to amplification was between 32.33-65.33 minutes for these five kits. Conclusion The five fast nucleic acid detection reagents have good performance and can be used as a supplement to routine nucleic acid detection reagents. © 2022 Chinese Journal of Laboratory Medicine. All rights reserved.

Wastewater-Based Epidemiology: Detection of SARS-CoV-2 RNA in Different Stages of Domestic Wastewater Treatment in Santa Fe, Argentina.

The COVID-19 pandemic affected human life at every level. In this study, we analyzed genetic markers (N and ORF1ab, RNA genes) of SARS-CoV-2 in domestic wastewaters (DWW) in San Justo City (Santa Fe, Argentina), using reverse transcription-quantitative real-time PCR. Out of the 30 analyzed samples, 30% were positive for SARS-CoV-2 RNA. Of the total positive samples, 77% correspond to untreated DWW, 23% to pre-chlorination, and no SARS-CoV-2 RNA was registered at the post-chlorination sampling site. The viral loads of N and OFR1ab genes decreased significantly along the treatment process, and the increase in the number of viral copies of the N gene could anticipate, by 6 days, the number of clinical cases in the population. The concentration of chlorine recommended by the WHO (≥ 0.5 mg L-1 after at least 30 min of contact time at pH 8.0) successfully removed SARS-CoV-2 RNA from DWW. The efficiency of wastewater-based epidemiology (WBE) confirms the need to control and increase DWW treatment systems on a regional and global scale. This work could contribute to building a network for WBE to monitor SARS-CoV-2 in wastewaters during the pandemic waves and the epidemic remission phase. Supplementary Information: The online version contains supplementary material available at 10.1007/s11270-022-05772-w.

Establishment and evaluation of a quadruple quantitative real-time PCR assay for simultaneous detection of human coronavirus subtypes.

BACKGROUND: The newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and four seasonal human coronaviruses (HCoVs) (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1) still circulate worldwide. The early clinical symptoms of SARS-CoV-2 and seasonal HCoV infections are similar, so rapid and accurate identification of the subtypes of HCoVs is crucial for early diagnosis, early treatment, prevention and control of these infections. However, current multiplex molecular diagnostic techniques for HCoV subtypes including SARS-CoV-2 are limited. METHODS: We designed primers and probes specific for the S and N genes of SARS-CoV-2, the N gene of severe acute respiratory syndrome coronavirus (SARS-CoV), and the ORF1ab gene of four seasonal HCoVs, as well as the human B2M gene product. We developed and optimized a quadruple quantitative real-time PCR assay (qq-PCR) for simultaneous detection of SARS-CoV-2, SARS-CoV and four seasonal HCoVs. This assay was further tested for specificity and sensitivity, and validated using 184 clinical samples. RESULTS: The limit of detection of the qq-PCR assay was in the range 2.5 × 101 to 6.5 × 101 copies/µL for each gene and no cross-reactivity with other common respiratory viruses was observed. The intra-assay and inter-assay coefficients of variation were 0.5-2%. The qq-PCR assay had a 91.9% sensitivity and 100.0% specificity for SARS-CoV-2 and a 95.7% sensitivity and 100% specificity for seasonal HCoVs, using the approved commercial kits as the reference. Compared to the commercial kits, total detection consistency was 98.4% (181/184) for SARS-CoV-2 and 98.6% (142/144) for seasonal HCoVs. CONCLUSION: With the advantages of sensitivity, specificity, rapid detection, cost-effectiveness, and convenience, this qq-PCR assay has potential for clinical use for rapid discrimination between SARS-CoV-2, SARS-CoV and seasonal HCoVs.

Development of Quantitative Real-Time PCR for Detecting Environmental DNA Derived from Marine Macrophytes and Its Application to a Field Survey in Hiroshima Bay, Japan

The sequestration and storage of carbon dioxide by marine macrophytes is called blue carbon;this ecosystem function of coastal marine ecosystems constitutes an important countermeasure to global climate change. The contribution of marine macrophytes to blue carbon requires a detailed examination of the organic carbon stock released by these macrophytes. Here, we introduce a quantitative real-time polymerase chain reaction (qPCR)-based environmental DNA (eDNA) system for the species-specific detection of marine macrophytes. and report its application in a field survey in Hiroshima Bay, Japan. A method of qPCR-based quantification was developed for mangrove, seagrass, Phaeophyceae, Rhodophyta and Chlorophyta species, or species-complex, collected from the Japanese coast to investigate their dynamics after they wither and die in the marine environment. A trial of the designed qPCR system was conducted using sediment samples from Hiroshima Bay. Ulva spp. were abundant in coastal areas of the bay, yet their eDNA in the sediments was scarce. In contrast, Zostera marina and the Sargassum subgenus Bactrophycus spp. were found at various sites in the bay, and high amounts of their eDNA were detected in the sediments. These results suggest that the fate of macrophyte-derived organic carbon after death varies among species.

Diagnostic evaluation of qRT-PCR-based kit and dPCR-based kit for COVID-19.

BACKGROUND: Coronavirus disease of 2019 (COVID-19) is well known as a fatal disease, first discovered at Wuhan in China, ranging from mild to death, such as shortness of breath and fever. Early diagnosis of COVID-19 is a crucial point in preventing global prevalence. OBJECTIVE: We aimed to evaluate the diagnostic competency and efficiency with the Allplex™ 2019-nCoV Assay kit and the Dr. PCR 20 K COVID-19 Detection kit, designed based on the qRT-PCR and dPCR technologies, respectively. METHODS: A total of 30 negative and 20 COVID-19 positive specimens were assigned to the diagnostic test by using different COVID-19 diagnosis kits. Diagnostic accuracy was measured by statistical testing with sensitivity, specificity, and co-efficiency calculations. RESULTS: Comparing both diagnostic kits, we confirmed that the diagnostic results of 30 negative and 20 positive cases were the same pre-diagnostic results. The diagnostic statistics test results were perfectly matched with value (1). Cohen's Kappa coefficient was demonstrated that the given kits in two different ways were "almost perfect" with value (1). In evaluating the detection capability, the dilutional linearity experiments substantiate that the Dr. PCR 20 K COVID-19 Detection kit could detect SARS-CoV-2 viral load at a concentration ten times lower than that of the Allplex™ 2019-nCoV Assay kit. CONCLUSIONS: In this study, we propose that the dPCR diagnosis using LOAA dPCR could be a powerful method for COVID-19 point-of-care tests requiring immediate diagnosis in a limited time and space through the advantages of relatively low sample concentration and small equipment size compared to conventional qRT-PCR.