ABSTRACT
Objective To investigate the impact of pooling of inactivated samples on testing results of nucleic acid detection of SARS-CoV-2, and to provide a scientific detection scheme for the SARS-CoV-2 nucleic acid screening of large population samples. Methods The SARS-CoV-2 positive throat swab samples and the negative throat swab samples were inactivated at 56°C for 30 minutes, and mixed according to the ratio of positive samples to negative samples at 1:4, 1:9, and 1:19, respectively. Real-time fluorescent RT-PCR technology was used to detect the ORF-lab and N genes in the original solution and mixed solution. Results The nucleic acid test results of the 20 groups of inactivated samples were all positive. The nucleic acid test results of the 1:5 mixture and the 1:10 mixture were also positive. One group of samples of the 1:20 mixture was negative for ORF-lab and positive for the N gene. The Ct values of nucleic acid detection among all groups were significantly correlated. There was no statistically significant difference in the positive rate between different sample groups. Compared with the original solution, the Ct values of the ORF-lab gene of 1:5 mixture, 1:10 mixture, and 1:20 mixture samples increased by 1.73, 2.86, and 4.05, respectively, while the Ct values of the N gene of 1:5 mixture, 1:10 mixture and 1:20 mixture samples increased by 1.69, 2.79, and 3.25, respectively. Conclusion When conducting nucleic acid screening for SARS-CoV-2 in large population samples, a mixed test of less than 10 inactivated samples would not affect the qualitative results in most cases, but the results of weak positive samples may be affected.
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Objective To understand the status of new coronavirus contamination in the biosafety laboratory environment, identify key areas prone to contamination, and provide evidence for disinfection of central objects. Methods surfaces of high-frequency contact environment and protective equipment were sampled with moistened sterile cotton swabs after experiment and before disinfection, the results of the one-step real-time reverse transcription polymerase chain reaction (RT-PCR) of open reading frame 1ab and N fragment were used to evaluate the pollution status. Results Environmental surveys found 4 of 217 samples of environmental objects to be positive for new coronavirus RNA, that positive rate was 1.84%. Among them, BSL-3, BSL-2, and BSL-1 were sampled 23, 184, and 10 respectively. The 3 positive samples were from surfaces of nucleic acid extraction room of BSL-2 and from the handles of pass-through box, laboratory door handles and the outer surface of the alcohol watering pot respectively. The 1 positive sample was from the forearm of the protective clothing in BSL-2 laboratory. Conclusion There was a certain degree of virus pollution in key areas of the new coronavirus laboratory. The BSL-2 laboratory has a higher risk of environmental pollution than the BSL-3 and BSL-1 laboratories.
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A total of 100 HIN1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang,Hubei and Guangdong between June and November 2009,were provided by local CDC laboratories.After MDCK cell culture,57 Influenza A Pandemic (H1N1) viruses were isolated and submitted for whole genome sequencing.A total of 39 HA sequences,52 NA sequences,36 PB2 sequences,31 PB1 sequences,40 PA sequences,48 NP sequences,51 MP sequences and 36 NS sequences were obtained,including 20 whole genome sequences.Sequence comparison revealed they shared a high degree of homology (96％～99％) with known epidemic strains (A/Califomia/04/2009(H1N1).Phylogenetic analysis showed that although the sequences were highly conserved,they clustered into a small number of groups with only a few distinct strains.Site analysis revealed three substitutions at loop 220 (221-228) of the HA receptor binding site in the 39 HA sequences:A/Hubei/86/2009 PKVRDQEG→PKVRDQEA,A/Zhejiang/08/2009 PKVRDQEG→PKVRDQER,A/Hubei/75/2009PKVRDQEG→PKVRDQGG,the A/Hubei/75/2009 was isolated from an acute case,while the other two were from patients with mild symptoms.Other key sites such as 119,274,292 and 294 amino acids of NA protein,627 of PB2 protein were conserved.Meanwhile,all the M2 protein sequences possessed the Ser32Asn mutation,suggesting that these viruses were resistant to adamantanes.Comparison of these sequences with other H1N1 viruses collected from the NCBI database provides insight into H1N1 transmission and circulation patterns.
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Objective:To explore a rapid, economical and efficient approach for molecular detection of 22q11.2 micro-deletion syndrome. Methods: Fifty ventricular septal defect (VSD) patients (33 males and 17 females, age ranged from 1 month to 15 years), who were hospitalized in Nanjing Children's Hospital from Jan. 2004 to Jan. 2005, were randomly selected for this study. The peripheral blood of VSD patients and the buckle cells of their parents were obtained. Three short tandem-repeat polymorphism (STRP) markers, D22S944, 22D_4_2 and 22D_4_3, were used for fluorescent in situ hybridization(FISH)study and genotype analysis. Results: 22D_4_2 and 22D_4_3 produced clear electrophoresis band, and the detections were rapid and efficient. The 3 STRP markers were consistent with the Hardy-Weinberg equilibrium expectations, and their heterozygosity was high in the present population from Chinese Han nationality in Jiangsu province. FISH confirmed that 5 of the 50 VSD patients had a deletion within chromosome 22q11.2. Conclusion: Three STRP markers (D22S944, 22D_4_2 and 22D_4_3) (analysis) combined with FISH as a supplementary is an efficient and reliable approach for detection of (22q11.2 microdeletion.)