Meta-Analysis Of Detection Methods For Staphylococcus Aureus And Construction And Evaluation Of A New Immunological Detection Method

Objective:Aiming at the current commercially available S.aureus detection reagents,this study systematically evaluates the diagnostic value of each detection method by meta-analysis,and discusses the factors that affect the diagnostic performance of each method.In addition,in view of the deficiency of on-site detection of S.aureus,a new colorimetric immunodetection based on nano-materials was constructed by using the principle of immunological detection technology,and the practicability and diagnostic efficiency of this method were evaluated.Method:Pub Med,MEDLINE and Embase were searched for published English articles on the diagnosis of S.aureus infection by PCR,chromogenic media and immunological detection reagents.The search period is from January 1,2010 to September 31,2021.The articles were screened according to the pre-established inclusion and exclusion criteria,and the obtained data were analyzed using STATA software.Sensitivity,specificity,diagnostic odds ratio（DOR）and area under curve were calculated.Publication bias,heterogeneity test,and sensitivity analysis were performed to evaluate the reliability and robustness of the results.Meta regression and subgroup analysis were performed with the test object,sample source,target gene,incubation time,publication year,and sample size as covariates.Three kinds of nano-materials including double-labeled nano-probe（Ig Y-Au NP-ALP）,non-specific magnetic beads（n MBs）and gold nanorods were prepared,and their morphology and size were characterized.The n MBs were used as separator to capture S.aureus,and Ig Y-Au NP-ALP was used to specifically bind to the target bacteria.To construct a rapid detection method for S.aureus based on color reaction catalyzed by ALP.The experimental conditions were optimized,and the detection limit,specificity and the feasibility of the established method in practical application were evaluated.Results:1.The combined sensitivity,specificity and DOR of PCR were 92.6%,98.1%and 644.16,respectively.The differences in sensitivity and specificity of PCR among different test subjects,sample sources,target genes,publication year and sample size were statistically significant（P<0.001）,and only the differences in specificity among different cycle thresholds were statistically significant（P<0.001）.The combined sensitivity,specificity and DOR of chromogenic medias were 94.8%,98.7%and1523.28,respectively.There were significant differences in sensitivity and specificity between different sample sources,different incubation times and different sample sizes（P<0.001）.The combined sensitivity and specificity of immunological detection technology were 96.9%and 99.9%,respectively.2.The diagnostic value between PCR and chromogenic medias were compared with the detection of nasal swabs and methicillin-resistant S.aureus（MRSA）.For nasal swabs,the sensitivity,specificity and DOR of PCR were 92.1%,97.2%and 414.47,respectively,while the sensitivity and specificity of chromogenic medias were 91.4%,97.5%and 439.75,respectively.The differences in sensitivity and specificity were statistically significant（P<0.001）.For MRSA testing,the sensitivity,specificity and DOR of PCR were 91.0%,98.1%and 539.99,respectively,while the sensitivity,specificity and DOR of chromogenic medias were 94.9%,98.8%and 1660.75,respectively.The differences in sensitivity and specificity were statistically significant（P<0.001）.3.Ig Y-Au NP-ALP showed a spherical shape with good dispersion,uniform particle size and a diameter of about 20±2 nm.The ultraviolet spectrum showed a characteristic absorption peak at 526 nm,and the infrared spectrum showed characteristic peaks such as-CH₂-and C=O.The surface potential of Ig Y-Au NP-ALP was-5.7±1.2 m V.Ig Y-Au NP-ALP showed the enzymatic activity of catalyzing AAP to produce color reaction.The n MBs were spherical with an average particle size of 14±3 nm and showed good superparamagnetic properties.The gold nanorods presented a rod-like structure with good dispersion,and their longitudinal length was about 45nm and the transverse diameter was about 10 nm.The solution of gold nanorods was pink,and the transverse and longitudinal maximum absorption peaks of gold nanorods were located at 520 nm and 764 nm.4.After the optimization of experimental conditions,the p H of Ig Y-Au NP-ALP was 9.5,the co-incubation time of Ig Y-Au NP-ALP with magnetic complex was 30min,the enzymatic action time of ALP was 30 min,and the AAP concentration was 20m M.5.The detection limit of this method is 10³ CFU/m L by visual observation,with7 CFU/m L by quantitative analysis of UV spectrum.The detection of interfering bacteria proved that this method had excellent specificity.The recovery rate of this method in simulated milk samples was 97.3%-102.1%.Compared with the national standard method,the sensitivity,specificity and the Kappa value of this method were100%,80%,0.865,respectively.The results of Passing-Bablok regression and Bland-Altman plot showed that this method had good consistency with the national standard method,showing strong anti-interference ability in practical application.Conclusion:It was confirmed that the existing three types of detection methods,including PCR,chromogenic medias and immunological methods,all had good diagnostic value by using meta-analysis and calculating the effect size such as combined sensitivity.Among the three methods,immunological methods had the best diagnostic accuracy,and the chromogenic medias had higher diagnostic value than PCR in the detection of nasal swabs and MRSA.Based on meta regression and subgroup analysis,it was speculated that sample source,cycle threshold,detection gene and incubation time were the influencing factors of diagnostic value.The established immunocolorimetric detection method based on three nanomaterials,including Ig Y-Au NP-ALP,n MBs,and gold nanorods,has a lower limit of detection and good specificity in the detection of S.aureus.This method has great anti-interference ability in the food samples detection and good consistency with the national standard method,which is suitable for screening and on-site detection of S.aureus.