Establishment of a one-step qRT-PCR assay for detecting milRNA encoded by novel Bunyavirus and its clinical application value
10.13602/j.cnki.jcls.2025.07.10
- VernacularTitle:新布尼亚病毒编码milRNA qRT-PCR检测一步法的建立及其临床应用价值
- Author:
Hanying WU
1
;
Yuan FANG
;
Xi CHEN
;
Ping YANG
Author Information
1. 南京医科大学公共卫生学院,南京 211100
- Publication Type:Journal Article
- Keywords:
microRNA-like small RNAs;
one-step qRT-PCR;
novel Bunyavirus
- From:
Chinese Journal of Clinical Laboratory Science
2025;43(7):534-540
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish an efficient and stable one-step real-time quantitative PCR(qRT-PCR)method for detecting mi-croRNA-like small RNAs(milRNAs)encoded by novel Bunyavirus and evaluate its clinical application value.Methods Three kinds of milRNAs encoded by novel Bunyavirus,including sRNA S-1480,sRNA M-692,and sRNA L-4706,were selected based on prelimi-nary screening.The specific qRT-PCR primers and reaction systems for them were designed and optimized.The sensitivity,specificity,and clinical applicability of the method were further evaluated by standard curve drawing,primer concentration/sequence optimization,fluorescent dye optimization,product sequencing verification,and serum specimen detection of 20 patients infected with novel Bunya-virus and 20 healthy controls.Results A one-step qRT-PCR detection system was successfully established.The standard curve con-structed with standard substance showed a good linear relationship in the range of 10 fmol/L to 100 nmol/L(R2>0.98).The detection limits for three kinds of milRNAs were 10 fmol/L.The analysis results of the receiver operating characteristics(ROC)curve showed that sRNA S-1480(AUCROC=0.972 5),sRNA M-692(AUCROC=0.757 5),sRNA L-4706(AUCROC=0.957 5),and their combina-tion(AUCROC=0.995 0)could effectively distinguish the patients infected with novel Bunyavirus from healthy controls.Conclusion The established one-step qRT-PCR detection system exhibits high sensitivity and provides an efficient and reliable alternative for the clinical diagnosis of novel Bunyavirus infection.
