Standardized Operation Management Of Nucleic Acid Testing Laboratory Comparison Of Sensitivity Of Different Nucleic Acid Test Kits

The COVID-19 pandemic has enhanced the global capacity to respond to public health emergencies and is a wake-up call for all of humanity to fight and prevent pandemic diseases.A complete pathogen early warning and response system is a strategic reserve for national defense against biological threats.Rapid defense against emerging infectious agents determines whether humans can respond quickly and early to the next pandemic.This paper explores and establishes a set of biosafety Level II laboratory,including pathogen nucleic acid detection,gene sequencing and rapid diagnosis system,and its application in the prevention and control of novel coronavirus outbreak.Firstly,the establishment of biosafety secondary experimental system documents was carried out.The system documents of laboratory operation and management standards have been formulated according to the requirements of CNAS-CL02-A009"Application of Criteria for Accreditation of Medical Laboratory Quality and Competence in the Field of Molecular Diagnostics",CNAS-GL039"Guidance for Performance Verification of Molecular Diagnostic Testing Procedures"and CLSI-MM03.Including procedure documents,quality manual,risk assessment and standard operation,and carried out the laboratory daily operation,standard operation,personnel management,biosafety risk and other aspects of the discussion.During the prevention and control period of novel coronavirus,the company passed the evaluation and obtained the qualification for nucleic acid detection and confirmation of novel coronavirus,influenza virus and new emergent pathogens.During the prevention and control period of the novel coronavirus epidemic,7,910 nucleic acid tests were ensured and served as the scientific support platform of nucleic acid testing for the novel coronavirus related scientific research of the unit.Secondly,in order to ensure the operation and experimental consistency of standard laboratories,this paper formulated and standardized the novel coronavirus operating standards and completed the related risk assessment.The applicability of the novel coronavirus nucleic acid test kits widely used in the market was compared under the standardized operation management.Through the requirements of testing time,configuration steps,matching consumables and other requirements of the kit,the detection kit that can meet the main requirements of the laboratory for scientific research is screened and obtained.The kit requires four steps to complete the system preparation,and can complete the sample detection in 1.22h.The kit can match a wide range of extraction reagents without special requirements,and the adaptive instrument is in line with the specific conditions of the laboratory.According to the operating procedure of the kit,the standard procedure for nucleic acid detection of novel coronavirus in our laboratory has been developed.At the same time,we explored and established a method to screen the standard test kit,which laid a foundation for our laboratory to engage in other pathogens and meet the standard requirements.The degree of morbidity in patients with novel coronavirus can be judged comprehensively by clinical symptoms and various indicators.However,whether it has become a source of infection or a spreader cannot be directly judged by clinical indicators.Fluorescence quantitative detection of CT value is the most intuitive index to determine whether the detox period.This study explored the relationship between the CT value of ORF1ab gene and virus content and isolation by fluorescence quantitative determination of novel coronavirus.Using TCID₅₀ as the base unit,the classical strain of SARS-CoV-2 was calibrated,and it was determined that when the CT value of ORF1ab gene was 23.49 and the CT value of N gene was 21.91,the virus content was equivalent to 100TCID₅₀,and it was capable of infusing Vero cell lines.When ORF1ab gene CT value was 33.69 and N gene CT value was 31.67,the virus content was equivalent to 10-2TCID₅₀,which did not have the ability to infect Vero cell lines.A total of 193 ORF1ab positive animal tissue samples were collected and grouped according to CT value.When ORF1ab gene CT value was 25.49 and N gene CT value was 24.31,100%had the ability to be infected with Vero cell line and were at high risk.When ORF1ab gene CT value was 28.42 and N gene CT value was 27.21,66%had the ability to infect Vero cell lines,and were at moderate risk.When ORF1ab gene CT value was 34.08,N gene CT value was 32.45,they did not have the ability to infect Vero cell line,and the transmission risk was low,which provided the basis for clinical judgment of detoxification and transmission risk.At the same time,ORF1ab gene CT value of 28.78,N gene CT value of29.72 can be used for virus isolation,to provide data support for scientific research.This study established the hardware equipment layout and software operation management specifications for the biosafety Level II laboratory for scientific research,emergency detection and confirmation of new emergent pathogens.On the basis of strict compliance with the operation management specifications,it has made outstanding contributions to the basic scientific research of novel coronavirus and the prevention and control of novel coronavirus.The test task was successfully completed,the standard process was developed for the test platform to undertake the emergency detection task and the strategic reserve was made for the next pandemic detection.