| Trichinella spiralis(T.spiralis)is a zoonotic food-borne parasite.Humans are infected by eating raw or undercooked meat that contains the infective larvae of T.spiralis.A disease caused by infection with T.spiralis is called trichinellosis.The disease distributes worldwide and seriously threatens human health and public safety.Therefore,a test for T.spiralis is a mandatory item in the slaughter of pigs.However,the present detection methods are deficient in daily monitoring for T.spiralis in pigs and modernized slaughter and processing.Therefore,the development of a rapid,sensitive,and on-site detection method for T.spiralis in modernized breeding and slaughterhouses is of great significance to prevent trichinellosis.The study based on surface-enhanced Raman spectroscopy(SERS)and upconversion phosphor immunochromatography assay(UCP-ICA)established a rapid detection system for T.spiralis,which is suitable for modernized breeding and slaughterhouses,and the contents and results are as follows:1.Establishment of the detection method of T.spiralis based on SERS.Colloid silver was prepared by hydroxylamine hydrochloride reducing silver nitrate,and its performance in Raman spectrum enhancement and interference on experimental samples was tested.20 Wistar rats were orally infected with T.spiralis muscle larvae(3500 T.spiralis ML per rat),and 20 Wistar rats consist of a parallel control group.After collecting the serum samples of two groups,the SERS spectra were obtained for the serum between uninfected T.spiralis and 28 days post infected(dpi)by a laser confocal Raman microscope.To explore the difference of spectra between the two groups,fluorescence background and area normalization in the data were preprocessed to generate comparable SERS spectra.Then,the preprocessed SERS spectra were analyzed and modeled by multivariate statistical analysis methods including principal component analysis(PCA)and linear discriminant analysis(LDA).The prepared colloid silver nanoparticles have a diameter of approximately 25 nm,homogeneous size,high purity,and good Raman spectrum enhancement effect.And the results of the study showed that the SERS spectra of serum were not significantly different between the two groups before infection.In contrast,the SERS spectra on 28 dpi were significantly different between the two groups.Furthermore,PCA combined with LDA was used to construct a diagnostic algorithm,and the sensitivity,specificity,and accuracy of the algorithm are 87.5 %,94.7 %,and 91.4 %,respectively.In summary,combined with PCA and LDA,the SERS spectra stimulated by colloid silver nanoparticles mixed serum is hopeful to applied in the detection of large-scale,on-site,rapid,label-free,high accuracy,which is great potential to apply in the prevention of trichinellosis.2.Establishment of detection method for T.spiralis used UCP-ICA technology.Excretorysecretory antigens(ES)and goat anti-rabbit Ig G were covalently coupled with the upconversion nanoparticles(UCNPs),respectively,and goat anti-swine Ig G(800ng/stripe)and rabbit anti-goat Ig G(200ng/ stripe)were used as test line(T line)and control line(C line),respectively,which was sprayed on Nitrate cellulose sheet(NC sheet).The method was named T.spiralis upconversion phosphor immunochromatography assay(Ts-UCP-ICA).After optimized,the optimum serum dilution is 1:150,and the optimum sample treatment solution of the method is 100 m M HEPES p H 7.5,270 m M Na Cl,0.5 % v/v Tween-20,1 % v/v BSA.169 negative serums were tested by this method.According to the test results,a Low specificity cut-off value and High specificity cut-off of Ts-UCP-ICA were 0.1906(T/C ratio)and0.3233,respectively.In addition,serum samples from pigs infected with Cysticerci of Taenia asiatica,Cysticerci of Taenia solium,or Toxoplasma gondii were tested by this method,and the results showed that this method was remarkable specificity and no cross-reaction.The serum of pigs infected with 100,1000,and 10000 ML was tested,and the results indicated that the serum of pigs infected with those three doses could be detected positive result on the 35,30,and 25 dpi.Single-blinded assay of 55 swine serum found that the method has a specificity of 100 % and a sensitivity of 100 % in a Low specificity cut-off,and a specificity of 100 % and a sensitivity of80 % in High specificity cut-off.In addition,the coincidence of the method with the artificial digestion method is 87.27 %,and a value of Kappa is 0.7454.The materials of the method are valid until six months in 4 ℃.In summary,the two established detection methods in the study constitute a rapid detection system for T.spiralis.The method based on SERS possesses some characters,including rapid and high throughput,which is suitable for screening in large-scale samples.And the method based on UCP-ICA can be used for further determination in some unconfident serum samples from the first method.The system can promote an efficiency and an effect in the detection of T.spiralis and is also worthwhile in an epidemiological survey for trichinellosis and point-of-care test in the breeding and slaughterhouses,which would provide a technological support to prevent the disease. |
