Résumé
To develop a multiplex fluorescent quantitative RT-PCR for the detection of porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and swine acute diarrhea syndrome coronavirus (SADS-CoV), in this study, specific primers/probes were designed based on the conserved regions of M, M and N gene sequences of PEDV, PDCoV and SADS-CoV, respectively. After optimization of the reaction conditions, a multiplex fluorescent quantitative RT-PCR for PEDV, PDCoV and SADS-CoV was established. The results of specificity assay showed that the method was positive for detection of PEDV, PDCoV and SADS-CoV, and negative for detection of porcine transmissible gastroenteritis virus, porcine rotavirus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine circovirus type 2, porcine parvovirus, classical swine fever virus and foot-and-mouth disease virus. The results of sensitivity assay showed that the detection limit of this method for PEDV, PDCoV, and SADS-CoV plasmids standard was 1.0x101 copies/L, and had a good linear relationship with their Ct values in the range of 101 copies/L to 106 copies/L. The results of repeatability assay showed that the coefficients of variation (CVs) of intra- and inter-assay reproducibility ranged from 0.33% to 2.53%, indicating good repeatability and stability. To evaluate the effects of the developed method, 100 clinical samples collected from different parts of Henan province were used for detection of these three viruses and compared with those of single RT-PCR and standard methods. The results of multiplex fluorescent quantitative RT-PCR showed that the positive rates of PEDV, PDCoV and SADS-CoV were 38% (38/100), 14% (14/100) and 5% (5/100), respectively. There was no mixed infection. The coincidence rate with the standard detection methods of PEDV and PDCoV was 100%, and the sensitivity was higher than that of single RT-PCR. In this study, a specific, sensitive and rapid multiplex fluorescent quantitative RTPCR method was established for the first time, which could be used for the differential detection of PEDV, PDCoV and SADS-CoV, and laid a foundation for the differential diagnosis and control of porcine diarrheal diseases.
Résumé
OBJECTIVE: To explore a new method for detecting respiratory viruses by extracting residual virus on mask, and verify its reliability and sensitivity. METHODS: The novel coronavirus analogs-s La Sota strains of chicken Newcastle disease virus and H120 strains of infectious bronchitis virus with different diluted concentrations were sprayed onto surgical masks and N95 masks through a respiratory simulator, and they were left standing at room temperature for 2 hours and 12 hours, respectively. The cDNA and its amplification cycle(CT) values of the nucleoocapsid protein(N) of chicken Newcastle disease virus and the nucleoprotein(NP) genes of infectious bronchitis virus were detected by ordinary polymerase chain reaction(PCR) and quantitative real-time PCR(qRT-PCR). The minimum detectable virus concentration and viral content in masks under different retention times were compared. RESULTS: The gene bands of the Newcastle disease virus La Sota strains and the infectious bronchitis virus H120 strains were detected on the masks stored for different times, and the total RNA of the virus had good amplification curves in the range of 10 pg-10 ng. The mean CT values of N gene and NP gene of the residual virus on the general medical surgical mask and N95 masks placed for 2 h were 22.547+or-0.342,23.698+or-0.501 and 22.855+or-0.308,24.036+or-0.338, respectively. However, only part of them could be detected after 12 h. respectively, and there was no significant difference in CT values between the two masks during the same period of time(P2 h=0.452, P12 h=0.355). The minimum detectable concentration of virus in the masks was 1:800, and the number of residual viruses on the mask that can be detected was 6.75x10~3. CONCLUSION: The method of screening coronavirus by detecting virus residues on masks within 2 hours was feasible and suitable for medical surgical masks and N95 masks, which can be used for preliminary screening of respiratory viruses.