Coronavirus Nucleic Acid Detection Research Articles

Development of a low-cost multi-channel nucleic acid detection PCR instrument and clinical detection application of COVID-19

Since the COVID-19 outbreak at the end of December 2019, a variety of novel Coronavirus nucleic acid detection methods have been proposed at home and abroad. Because of the disadvantages of most existing PCR instruments on the market such as long reaction time and high cost, this study developed a more timesaving and cheaper two-channel real-time quantitative PCR instrument. In this instrument, a PCR system combining a thermal cycle system and real-time fluorescence quantitative technology was designed. The software system and data processing, optical system, thermal cycle module, and hardware module of the PCR instrument were studied. The low-cost, portable real-time quantitative PCR system has been validated with consistent results compared to Bio-rad CFX Connect. At the same time, the same samples were used for the contract experiment with the hospital instrument, and the amplification result was better than the existing instrument in the hospital.

Different Laboratory Diagnosis Methods of COVID-19: A Systematic Review

: The virus causing COVID-19 disease is known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disease spread rapidly and was transmitted like a contagious disease throughout China, and then it gradually spread in other parts of the world. Accordingly, the rapid and accurate detection of the SARS-CoV-2 virus plays an essential role in selecting timely treatments, saving lives, and preventing the spread of the disease. This study summarizes the methods used to identify coronavirus nucleic acid. The effectiveness of coronavirus nucleic acid detection kits by different samples and the performance of other diagnostic techniques are also addressed in this study. We searched Embase, Google Scholar, MEDLINE, Web of Science, Scopus, and PubMed databases as well as the references of all relevant articles in English published during 2019 - 2020 using keywords related to COVID-19, detection kits, and respiratory failure and proceedings from relevant conferences and congresses. The authors collected the relevant reports, and each of the authors independently reviewed the data published in different studies. The results of previous studies indicated that the diagnosis methods of the COVID-19 disease are the RT-PCR method, ELISA kits, quick tests, white blood cell count, C-reactive protein (CRP) levels, other laboratory factors and antigenic detection methods. Given the sensitivity and specificity of these methods at different periods using different samples, the disease interpretation can be performed accurately. The findings showed that proper laboratory equipment and appropriate laboratory kits are necessary for the rapid and precise identification of COVID-19.

Analysis of digestive endoscopic results during COVID-19

ObjectiveThis study aims to explore the necessity and safety of digestive endoscopy during the epidemic of coronavirus disease 2019.MethodsA retrospective cohort study method was used to collect patients’ data from the endoscopy center of the Civil Aviation General Hospital of China from February 1 to May 31, 2020, as the observation group. The patients’ data of endoscopic diagnosis and treatment during the same period in 2019 were used as a control group, to compare the differences in the number of diagnosis and treatment and the detection rate of gastrointestinal diseases in the two groups. At the same time, patients and related staff were followed up for the situation of new infection.ResultsDuring the epidemic, our endoscopy center conducted a total of 1,808 cases of endoscopic operations and 5,903 cases in the control group. The amount of endoscopic work during the epidemic period was 30.63% in the same period last year. During the epidemic, 26 patients underwent endoscopic mucosal resection (EMR)/endoscopic submucosal dissection (ESD) treatment, 26 patients underwent ERCP, and 18 patients underwent gastrointestinal stent implantation. In the control group, 273 patients underwent EMR/ESD, 17 underwent ERCP, and 16 underwent gastrointestinal stenting. During COVID-19, compared with the same period last year, the detection rates of peptic ulcer, esophageal cancer, gastric cancer, colon cancer, and rectal cancer were significantly higher (χ2 = 4.482, P = 0.034; χ2 = 5.223, P = 0.006; χ2 = 2.329, P = 0.041; χ2 = 8.755, P = 0.003; and χ2 = 5.136, P = 0.023). Through telephone follow-up, novel coronavirus nucleic acid detection and blood antibody detection, no patients or medical staff were infected with the novel coronavirus.ConclusionDuring COVID-19, the number of digestive endoscopic operations decreased significantly compared with the same period last year, but the detection rate of various diseases of the digestive tract increased significantly. On the basis of strict prevention and control, orderly recovery of endoscopic work is essential.

Performance evaluation of six coronavirus nucleic acid detection reagents and matching analysis with nucleic acid extraction reagents

Objective: To select nucleic acid extraction reagents and amplification reagents by comparing the minimum detection limit, amplification efficiency, specificity, accuracy, and matching analysis with 6 nucleic acid extraction reagents of 6 novel coronavirus (2019-nCoV) nucleic acid detection reagents and provide scientific basis for clinical diagnosis and treatment. Methods: The national-standard material of 2019-nCoV nuclear acids was diluted as ths six fradient concentrations. Then, the diluted samples were analyzed by using the six kinds of agents/kits, Guangzhou Daan (DA), Beijing Jinhao (JH), Wuhan Mingde (MD), Sichuan Mike (MK), Jiangsu Shuoshi (SS) and Shanghai Zhijiang (ZJ). The minimum detection limit, amplification efficiency, accuracy, specificity and other properties of these kits were examined. The positive control levels 1, 2 (low and medium concentration) were extracted by using the six nucleic acid extraction reagents (numbered a-f) with six 2019-nCoV detective kits to detect target genes of 2019-nCoV genome, and compared between different nucleic acid extraction reagents and different nucleic acid detection reagents. Match between the different nucleic acid extraction reagents detection reagents, using two-way analysis of variance. Results: The minimum detection limit of Nucleocapsid (N) gene of DA, JH, MD, SS reagent was 7.0×102 copies/ml.The minimum detection limit of open reading frame 1ab (ORF1ab) gene of JH, MD, SS, MK reagent was 9.39 ×102 copies/ml.The minimum detection limit of envelope (E) gene of MK, ZJ reagent was 5.03×102 copies/ml. SS reagent N gene amplification efficiency was 89%, ORF1ab gene amplification efficiency was 90%, was the highest among six reagents.a, b, c, d extraction reagents and 6 nucleic acid detection reagents match well, But a extraction reagent was more suitable for use with SS,MK and ZJ reagents (P<0.05), d extraction reagent was more suitable for MD,SS,DA,JH and MK nucleic acid detection reagents(P<0.05);e extraction reagent was more suitable for SS nucleic acid detection reagents(P<0.05), f extraction reagent was most suitable for DA, JH Nucleic acid detection reagents (P<0.05);e and f reagents were not suitable for use with MK and ZJ. Conclusion: The 6 detection reagents have good performance, and the appropriate extraction reagents and detection reagents should be used in conjunction with the actual conditions of the laboratory.

Comparative analysis of detection performance of novel coronavirus nucleic acid detection reagents.

Objective To evaluate the detection performance of novel coronavirus nucleic acid reagents (Maccura Biological Co.,Ltd,fluorescent PCR method),it is validated clinically and used the same kinds of kits for comparsion research.Methods Compared with novel coronavirus nucleic acid reagents (Shanghai Zhijiang Biological Co.,Ltd,fluorescent PCR method),the similar products of Maccura Biological Co.,Ltd was to be estimated.Pharyngeal swab specimens of 14 COVID-19 patients diagnosed by the CDC and 211 general admission patients from February 18 to 24 were taken into our study,and two kits were used for parallel testing novel coronavirus (ORF1ab/N/E gene).Results The positive rate and negative coincidence rate of Maccura detection kit and Zhijiang' were more than 99.5%.Relative deviation of ORF1ab gene and N gene were not more than ±16%.Conclusion Novel coronavirus nucleic acid detection reagents produced by Maccura Biological Co.,Ltd has good consistency with similar reagents approved for marketing,which can meet the needs of clinical testing. (English) [ABSTRACT FROM AUTHOR] 目的 对迈克生物股份有限公司的新型冠状病毒核酸试剂盒(荧光PCR法)进行临床验证,并 用同类试剂盒进行比对研究。方法 以迈克生物股份有限公司的研发生产的新型冠状病毒核酸检测试剂盒 (荧光PCR法)作为待考核试剂,以上海之江生物科技有限公司的同类产品作为比对试剂。分别对14例由疾控 中心确诊的新型冠状病毒肺炎患者和2020年2月18日至2月24日211例普通入院患者的咽拭子标本进行新 型冠状病毒(ORF1ab/N/E基因)检测,比较两种试剂检测检出率的一致性。结果 迈克新型冠状病毒核酸检 测试剂盒与之江新型冠状病毒核酸检测试剂盒的检测结果阳性率和阴性符合率均达99.5%以上,ORF1ab基 因和N 基因测定值相对偏差不超过±16%。结论 迈克新型冠状病毒核酸检测试剂盒与已获批准上市的同类 试剂检测结果一致性良好,可满足临床检测需求。 (Chinese) [ABSTRACT FROM AUTHOR] Copyright of Journal of Modern Medicine & Health is the property of Journal of Modern Medicine & Health and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Standardized sputum collection increases sputum sample collection rate for novel coronavirus nucleic acid detection

To evaluate the effect of standardized health education on the sputum specimen collection rate for nucleic acid detection of coronavirus disease 2019 (COVID-19). Two hundred and twenty-seven patients in fever clinics and isolation wards of Sir Run Run Shaw Hospital of Zhejiang University and 307 migrant workers returning to 5 enterprises in Shanghai from February 3 to March 14, 2020 were enrolled in the study. Through clarifying the procedures of collecting sputum specimens, making graphic/video health education materials, standardizing the contents and methods of health education, we conducted education to the subjects. The subject expectorated spontaneously or with medical assistance. For patients, the number of sampling attempts and sputum acquisition times were documented before and after the implementation of the standardized expectoration method; for the returning migrant employees in the enterprises, only the number of collected samples after the implementation of the standardized expectoration method were recorded. A total of 378 sputum samples were collected from 227 patients. The sputum sampling rates before and after the implementation of health education were 40.9%and 58.4%, respectively (P<0.01). A total of 304 sputum samples were obtained from 307 enterprise returnees, with a sample collection rate of 99.0%. The education for standardized sputum sample collection method can effectively increase the sputum collection rate.

Emergency Biosafety Management Practice in Laboratory of Shelter Hospital.

At the end of 2019, the novel coronavirus infection outbroke in Wuhan, Hubei Province. On Feb. 2, 2020, Wuhan, as the worst-hit region, began to build “shelter hospital” rapidly to treat patients with mild illness. The shelter hospital has multiple functions such as emergency treatment, surgical treatment and clinical test, which can adapt to emergency medical rescue tasks. Based on the characteristics that shelter hospital only treats patients with mild illness, tests of shelter laboratory, including coronavirus nucleic acid detection, IgM/IgG antibody serology detection, monitoring and auxiliary diagnosis and/or a required blood routine, urine routine, C-reactive protein, calcitonin original, biochemical indicators (liver enzymes, myocardial enzymes, renal function, etc.) and blood coagulation function test etc, were used to provide important basis for the diagnosis and treatment of the disease. In order to ensure laboratory biosafety, it is necessary to first evaluate the harm level of various specimens. In the laboratory biosafety management, the harm level assessment of microorganisms is the core work of biosafety, which is of great significance to guarantee biosafety. As an emergency deployment affected by the environment, shelter laboratory must possess strong mobility. This paper will explore how to combine the biosafety model of traditional laboratory with the particularity of shelter laboratory to carry out effective work in response to the current epidemic.

Emergency response to COVID‐19 epidemic: One Chinese blood centre's experience

ObjectiveThe COVID‐19 epidemic has caused a significant global social and economic impact since December 2019. The objective of this study was to demonstrate the emergency response of a Chinese blood centre on maintaining both the safety and the sufficiency of blood supply during large, emerging, infectious epidemics.Materials and MethodsEarly on in the outbreak of COVID‐19, the Chengdu Blood Center developed strategies and implemented a series of measures, including enhanced recruitment efforts, addition of new donation deferral criteria and notification after donation, optimisation of donor experience, development and implementation of a new coronavirus nucleic acid detection technology platform for blood screening and screening all donations for SARS‐CoV‐2 RNA to maximumly protect the safety of blood supply during a time of unclear risk.ResultsStarting on February 20, the immediate satisfaction rate of blood product orders in Chengdu city's clinical settings reached 100%, and there was no case of blood transfusion infection.ConclusionThe recent experience during the outbreak of SARS‐CoV‐2 reminded us that improvement in the areas of national and international collaborative programmes for dealing with blood availability and safety concerns during early stages of a disaster and regional and national mechanisms for timely communication with the general public on behalf of blood services should help to better prepare us for future disasters.

New molecular diagnostic technologies for clinical detection of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an ongoing pandemic of new coronavirus pneumonia (corona virus disease 2019, COVID-19). The virus has a long incubation period and strong infectivity, which poses a major threat to global health and safety. Detection of SARS-CoV-2 nucleic acid lies at the center of rapid detection of COVID-19, which is instrumental for mitigation of the ongoing pandemic. As of August 17, 2020, The National Medical Products Administration in China has approved 15 new coronavirus nucleic acid detection kits, 10 kits of which are based on reverse transcription-real-time quantitative PCR (RT-qPCR) technology. The remaining kits use five molecular diagnostic technologies different from RT-qPCR. This article reviews the principles, reaction time, advantages and disadvantages of above 15 detection kits, in order to provide references for rapid screening, diagnosis, prevention and control of COVID-19 and similar infectious diseases.

Clinical application effect of modified nasopharyngeal swab sampling for 2019 novel coronavirus nucleic acid detection

Objective: To study the clinical application effect of modified nasopharyngeal swab sampling for 2019 novel coronavirus nucleic acid detection. Methods: This study covered the period from January 14 to March 1, 2020.The supine position method and the protective face screen were used to collect nasopharyngeal swabs from February 24 onwards, before which, the nasopharyngeal swabs were collected by sitting position method. All the patients who were diagnosed with suspected/confirmed 2019 novel coronavirus infection were admitted from February 19 with the nasopharyngeal swabs collected outside the hospital before admission. (1) Thirty-four swabbing operators meeting the inclusion criteria of the study were recruited in this retrospective cohort study. They were grouped according to the collection method of nasopharyngeal swabs. Sixteen operators of Wuhan Taikang Tongji Hospital who applied the supine position method and the protective face screen were included in supine position method+ protective face screen group (15 males and 1 female, aged 34-49 years); 18 operators (12 from the First Affiliated Hospital of Army Medical University (the Third Military Medical University), 1 from Wuhan Jiangxia Mobile Cabin Hospital, 5 from the East District of People's Hospital of Wuhan University) who applied the traditional sitting position method were included in sitting position method group (2 males and 16 females, aged 25-49 years). In supine position method+ protective face screen group, when collecting sample, the patient lay flat and wore a special protective face screen for nasopharyngeal swab sampling, with neck slightly extending and face turning to the opposite side of the operator about 10°. The self-designed questionnaire was used to investigate the cooperation, the incidence of nausea, coughing, sneezing, and struggling of patients evaluated by the operators, the operation time for a single swab sample, the fear of operation and the perceived exposure risk of operators in the two groups. (2) Sixty-five patients (22 males and 43 females, aged 25-91 years) admitted to Wuhan Taikang Tongji Hospital who successively received the sitting position method and supine position method+ protective face screen for nasopharyngeal swabs sampling and with complete nucleic acid detection results were included. The positive rates of nucleic acid detection by the two sampling methods of nasopharyngeal swabs of the patients were statistically analyzed. (3) Forty-one patients who could express their feelings accurately were selected out of those 65 patients (12 males and 29 females, aged 27-83 years). The comfort of patients in the process of sampling by the two methods was investigated. (4) Thirty-four patients (10 males and 24 females, aged 25-83 years) with two or more consecutive negative results of nucleic acid detection of nasopharyngeal swabs by sitting position method were selected from the above 65 patients. The positive rate of nucleic acid detection of nasopharyngeal swab of patients by supine position method+ protective face screen, i. e. negative to positive rate was statistically analyzed. Data were statistically analyzed with t test, Wilcoxon signed rank test, and chi-square test. Results: (1) Compared with those of sitting position method group, the cooperation score of patients evaluated by the operators in supine position method+ protective face screen group was significantly higher (Z=-4.928, P<0.01), the incidence of nausea, choking cough, sneezing, and struggling of patients evaluated by the operators, and the fear of operation score and the perceived exposure risk score of operators in supine position method+ protective face screen group were significantly lower (Z=-5.071, -5.046, -4.095, -4.397, -4.174, -5.049, P<0.01), and the operation time for a single swab sample in supine position method+ protective face screen group was significantly longer (t=223.17, P<0.01). (2) The positive rate of nucleic acid detection of nasopharyngeal swabs by supine position method+ protective face screen was 60.00% (39/65), which was obviously higher than 41.54% (27/65) by sitting position method (χ(2)=4.432, P<0.05). (3) The comfort score of the 41 patients during nasopharyngeal swabs sampling by supine position method+ protective face screen was significantly higher than that by sitting position method (Z=-5.319, P<0.01). (4) Of the 34 patients with two or more consecutive negative results of nucleic acid detection of nasopharyngeal swabs by sitting position method, the rate of negative to positive of nucleic acid detection was 26.47% (9/34) after sampling by supine position method+ protective face screen. Conclusions: Compared with the traditional sitting position method, detection of 2019 novel coronavirus nucleic acids of nasopharyngeal swabs collected by supine method combined with protective face screen is worth promoting, because of its better comfort of patients, low exposure risk for operators, in addition to reducing in the false negative result to some extent, which may help reduce false recurrence of discharged patients.

Analysis of 8 274 cases of novel coronavirus nucleic acid detection and co-infection in Wuhan

Objective To investigate the positive rate for 2019-nCoV tests and co-infections in Wuhan district. Methods A total of 8 274 cases in Wuhan were enrolled in this cross-sectional study during January 20 to February 9, 2020, and were tested for 2019-nCoV using fluorescence quantitative PCR. Both respiratory tract samples (nasopharynx, oropharynx, sputum and alveolar lavage fluid) and non-respiratory tract samples (urine, feces, anal swabs, blood and conjunctival sac swabs) were collected. If both orf1ab and N genes are positive, they are classified as nucleic acid test positive group; if both orf1ab and N genes are negative, they are classified as negative group; if single gene target is positive, they are classified as suspicious group. Individuals were divided into male group and female group according to sex. At the same time, 316 patients were tested for 13 respiratory pathogens by multiplex PCR. Results Among the 8 274 subjects, 2 745 (33.2%) were 2019-nCoV infected; 5 277 (63.8%) subjects showed negative results in the 2019-nCoV nucleic acid test; and 252 cases (3.05%) was not definitive (inconclusive result). The age of cases with COVID-19 patients and inconclusive cases was significantly higher than that of cases without 2019-nCoV infection (40 vs 56, t=27.569, P<0.001; 52 vs 56, t=6.774, P<0.001). The positive rate of 13 respiratory pathogens multiple tests was significantly lower in 104 subjects who were positive for 2019-nCoV compared with those in subjects who were negative for 2019-nCoV test (5.77% vs 18.39%, χ2=24.105, P=0.003). Four types of respiratory tract samples and five types of non-respiratory tract samples were found to be positive for 2019-nCoV nucleic acid test. Conclusion The 2019-nCoV nucleic acid positive rate in male is higher than in female. Co-infections should be pay close attention in COVID-19 patients. 2019-nCoV nucleic acid can be detected in non-respiratory tract samples. Key words: Coronavirus; Severe acute respiratory syndrome; Reverse transcriptase polymerase chain Reaction; Virus diseases; Clinical laboratory techniques

The way to reduce the false negative results of 2019 novel coronavirus nucleic acid detection

核酸检测是新型冠状病毒肺炎(novel coronavirus pneumonia, NCP)确诊的重要手段，但是目前临床上反映存在较高比例核酸检测"假阴性"的问题。本文将明确核酸检测"假阴性"的概念，核酸检测为什么可以作为感染确诊"假阴性"，并分析造成核酸检测"假阴性"的原因。在核酸检测基础上，补充SARS-CoV-2病毒特异性IgM/IgG抗体的联合动态多次检测，可以明显减少有病毒性肺炎临床症状和影像学证据、但核酸多次或始终检测为阴性的临床层面上的"假阴性"，对临床诊断病例的最终确诊和准确性评价具有重要意义。.

The way to reduce the"false negative results"of 2019 novel coronavirus nucleic acid detection

核酸检测是新型冠状病毒肺炎(novel coronavirus pneumonia, NCP)确诊的重要手段，但是目前临床上反映存在较高比例核酸检测"假阴性"的问题。本文将明确核酸检测"假阴性"的概念，核酸检测为什么可以作为感染确诊"假阴性"，并分析造成核酸检测"假阴性"的原因。在核酸检测基础上，补充SARS-CoV-2病毒特异性IgM/IgG抗体的联合动态多次检测，可以明显减少有病毒性肺炎临床症状和影像学证据、但核酸多次或始终检测为阴性的临床层面上的"假阴性"，对临床诊断病例的最终确诊和准确性评价具有重要意义。.