Establishment of a national reference panel for Vibrio vulnificus nucleic acid detection kits
Objective To establish a national reference panel for Vibrio vulnificus nucleic acid de-tection reagents and set standards,which aimed to evaluate the quality of related kits.Methods A variety of Vibrio vulnificus and other Vibrio pathogens were collected and cultured.After colony identification,16s rRNA sequencing analysis and detection of a real-time fluorescence quantitative PCR reagent,18 samples were selected,diluted,and packaged to form a national reference panel for Vibrio vulnificus nucleic acid detection kits.Different laboratories were invited to coordinate calibration of the panel,and the uniformity and stability were further investigated.Results The established national reference panel included 8 positive references P1~P8,10 negative references N1~N10,1 repetitive reference R and 1 limited detection reference L;Refer-ence L was determined the concentration of 1×108 CFU/mL by colony-counting methods.Four laboratories par-ticipated in the collaborative calibration of national reference materials and developed quality standards based on the results:positive coincidence rate of 8/8,negative coincidence rate of 10/10;the detection limit is at least to be 1×103 CFU/mL,the repeatability is required to be positive for 10 times,and the variation coefficient of Ct value or quantitative value is not more than 5.0%.The coefficient of variation of Ct value in uniformity de-tection of a national reference was less than 5%,which met the requirements.The stability of the reference prod-uct was not affected when it was refrigerated at 2~8℃and room temperature for 7 days.Conclusion The nation-al reference panel for vibrio vulnificus nucleic acid detection kits was successfully established,which can be used for quality control and evaluation for enterprises which planned to develop and research related kits.