Establishment and application of RPA-LFD detection method for Pasteurella multocida from duck
10.16303/j.cnki.1005-4545.2025.03.10
- VernacularTitle:鸭源多杀性巴氏杆菌RPA-LFD检测方法的建立与应用
- Author:
Youci LONG
1
;
Qinglin GU
;
Simei XIAN
;
Weihao ZHENG
;
Qin WU
;
Mengyi YU
;
Jing LI
;
Shuaibin WU
Author Information
1. 贵州大学动物科学学院,贵州 贵阳 550025
- Publication Type:Journal Article
- Keywords:
Pasteurella multocida;
kmt1 gene;
RPA;
lateral flow dipstick
- From:
Chinese Journal of Veterinary Science
2025;45(3):466-472
- CountryChina
- Language:Chinese
-
Abstract:
This study aims to establish a convenient,new and visual detection method for the field diagnosis of Pasteurella multocida(Pm).With reference to the Pm kmt1 gene conserved sequence published in GenBank,PCR amplification primers were designed,the amplified kmt1 gene was cloned into pMD19-T vector,and the recombinant plasmid standard pMD19-T-kmt 1 was estab-lished and identified by PCR and sequencing.Using pMD1 9-T-kmt 1 plasmid as template and kmt1 gene as target gene,basic primers were designed and synthesized.According to the requirements of LFD,a probe(Pm-P)was designed,and the RPA-LFD method for Pm detection was established by optimizing the reaction conditions.Specificity and sensitivity tests were carried out,and 64 clini-cal samples were tested by the method.The results showed that the established Pm RPA-LFD method could be amplified at 37 ℃ for 15 min.Escherichia coli(E.coli),Salmonella enteriditis(SE),Riemerella anatipestifer(RA),Staphylococcus,goose parvovirus(GPV),duck plague virus(DPV),Muscovy duck parvovirus(MDPV)DNA was extracted as the template,and plasmid standard pMD19-T-kmt 1 was used as the positive control.All the positive controls were negative,indicating that the method had good specificity.The plasmid standard pMD1 9-T-kmt 1 was diluted with a 10-fold ratio,and the plasmid standard with a concentration of 107-100 copies/μL was used as the template.The sensitivity was 1.50×101 copies/μ,,which was 100 times higher than that of PCR.A total of 64 clinical samples with suspected RA were subjected to testing using PCR,RPA and LAMP-LFD,with a 100％compliance rate for all three detection tests.The results show that the established RPA-LFD method has the characteristics of strong specificity,high sensitivity,fast speed and visualization,and can be applied to the field detection of Pm.
