Establishment and application of the molecular-beacon-based asymmetric recombinase amplification for detecting Staphylococcus aureus
10.3760/cma.j.issn.1009-9158.2017.04.019
- VernacularTitle:不对称重组酶介导扩增结合分子信标检测金黄色葡萄球菌方法的建立与应用
- Author:
Lin ZHOU
;
Huan XU
;
Cheng YANG
;
Fengling ZHANG
;
Jie LUO
;
Wenbin JIANG
;
Chao WANG
;
Kai CHANG
;
Weiping LU
;
Ming CHEN
- Keywords:
Staphylococcus aureus;
Nucleic acid amplification techniques;
Molecular probes
- From:
Chinese Journal of Laboratory Medicine
2017;40(4):309-313
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a homothermal and fast detecting method on pathogenic bacteria by combining recombinase-aid amplification (RAA) with molecular beacon.Methods The establishment of the methodology.Staphylococcus aureus specific primers were designed from the relative region of the staphylococcal protein A (SPA).Asymmetry amplification was optimized by adjusting the primer concentration ratios.The results of amplification and hybridization were visualized and analyzed by agarose gel electrophoresis and fluorescence detection.The sensitivity was identified by detecting dilute positive plasmids.And the specificity was determined using RAA method by detecting 72 pathogenic bacteria,including Staphylococcus aureus and other Staphylococcus spp.from the Department of Clinical Laboratory of Daping Hospital in December 2016.Besides,the Kappa analysis and the clinical diagnosis efficiency were investigated by analyzing 39 extra strains in the laboratory in December 2016.Results When the concentration ratio of restrictive and non-restrictive primer was 1:20,the yield efficiency of single-stranded DNA (ssDNA) reached the peak.And as for the hybridization efficiency,the asymmetry amplification was higher than symmetry amplification.Twenty copies/μl was proposed as the limits of detection by testing dilute plasmids.And the RAA hybridization method could distinguish Staphylococcus aureus with other Staphylococcus spp.Comparing with traditional detection methods with a Kappa index of 0.860,this method shows a good consistency.By analyzing the 111 bacteria,the sensitivity of the method is 92.5％ (37/40),the specificity is 97.2％ (69/71),the positive predictive value is 94.9％ (37/39),the negative predictive value is 95.8％ (69/72),the positive likelihood radio is 33.04,the negative likelihood radio is 0.077,the Youden index is 0.897 and the Kappa index is 0.902.Conclusion Through the combination of asymmetry recombinase-aid amplification optimization and molecular beacon probe,a new method of detecting bacteria DNA with RAA hybridization technique is established,providing the foundation for its clinical application.
