免核酸提取快速检测技术在流感病毒细胞株鉴定中的应用

Application of Nucleic Acid Extraction Free Rapid Detection Technology in Identification of Influenza Virus Cell Lines

目的探索免核酸提取进行Real-time PCR在流感病毒细胞株中的应用。方法选取甲型H_1N_1、H_3N_2、BYamagata、B-Victorial 4种亚型流感病毒细胞株各10株,先进行血凝实验(Hemagglutination test,HA),再进行核酸提取与病毒液用Real-time PCR分别进行甲、乙、4种亚型核酸检测,比较其CT值差异。结果流感病毒HA几何平均滴度(GMT)和CT值的几何平均值无线性关系。甲、乙型核酸提取和不提取的CT值范围分别为10. 5～14. 7和10. 5～18. 2,甲型H1N1、H3N2、B-Yamagata、B-Victorial 4种亚型核酸提取和免提取的CT值范围分别为11. 4～16. 3和12. 0～16. 7; 11. 2～17. 2和10. 7～19. 7; 12. 5～16. 7和10. 3～16. 7; 11. 8～17. 1和12. 6～17. 2,各型别间CT值几何平均值无差异。结论免核酸提取在流感病毒分离时能快速检测病毒液CT值,缩短实验中间步骤。

Objective To investigate the application of Real - time PCR without nucleic acid extraction in identification of influenza virus cell lines. Methods Ten specimens of four subtypes of influenza virus cell lines including type A H1N1, H 3 N 2, B -Yamagata and B -Victorial were firstly detected by hemagglutination tests （HA） and processed by nucleic acid extraction, followed by assay of A, B and four subtypes by Real - time PCR. The cycle thresholds （CT） were compared. Results There was no linear relationship between the GMT of HA and the average value of CT. The CT ranges of A and B with and without nucleic acid extraction were 10.5 - 14. 7 and 10. 5 - 18.2, respectively. CT ranges of four subtypes including type A H1N1 , H3 N2, B - Yamagata and B -Victorial with and without nucleic acid extraction were 11.4 - 16. 3 and 12. 0 - 16. 7; 11.2 -17.2 and 10.7-19.7; 12.5-16.7 and 10.3-16.7; 12.5-16.7 and 10.7- 19.7, respectively. No difference was found between geometric means of CT of different types. Conclusion The nucleic acid extraction free technique allows rapid determination of CT values in isolation of influenza virus and can reduce experimental procedures.