| Sika deer antler is a valuable traditional chinese medicine,and deer blood contains a variety of bioactive substances,both of which have been used as medicinal or health tonics for thousands of years.The existence of contaminants will directly affect the quality of sika deer antler and deer blood related products,and bring huge hidden dangers to use safety.Therefore,it is very important to study and optimize the detection methods of contaminants residues in sika deer antler and deer blood.A real-time fluorescence quantitative PCR method for detecting brucella and mycobacterium tuberculosis in sika deer antler and deer blood was established.At the same time,based on the current situation of anesthetic residues caused by chemical guaranteed antler extraction technology,two veterinary anesthetics,celazazine and dimethylaniline thiazide,were determined by ultra-high performance liquid chromatography-triple quadruple-bar mass spectrometry and triple quadruple-bar gas chromatography-mass spectrometry.This paper is divided into four parts:(1)According to the reported specific gene sequence IS711 of brucella cervi,synthetic primers and fluorescent probes were designed and sent to the biological company.A real-time fluorescent quantitative PCR system for detection of brucella cervix was established using the successfully prepared standard as template.And the repeatability,specificity and sensitivity of the method were evaluated.The results showed that four groups of brucella IS711 positive plasmids were used as templates to complete the reaction under the same conditions.The Ct values were 15.13,16.58,16.17 and 17.09,respectively,which indicated that the method had good repeatability.The specificity of the system was investigated by using B.suis S2,B.abortus S19,B.melitensis M5,Mycobacterium tuberculosis H37Rv and BVDV NADL as templates.The results showed that mycobacterium tuberculosis H37Rv and BVDV NADL as templates had no obvious amplification curve,which could be judged as negative,indicating that the system had good specificity.The correlation coefficient R~2 of the standard curve was 0.996,showing a good linear correlation.Gradient dilution of positive markers with known concentration and minimum detection copy number of 4 Copies/uL showed that the method had good sensitivity.The established method was used to compare and validate the methods of suspected positive deer blood and fresh velvet antler samples.Of the 30 PPD positive deer blood samples,15 were positive.Among of the 30 PPD positive deer antler samples,15 were positive.A simple,rapid,sensitive and specific detection method was successfully constructed.(2)According to the gene sequence Rv3873 of Mycobacterium tuberculosis,specific primers and probes were designed and synthesized.The reaction conditions of the system were optimized by single factor test.A real-time fluorescence quantitative PCR method for Mycobacterium tuberculosis was established based on the successfully prepared standard sample.The experimental results showed that the constructed standard plasmids were used as templates to repeat four groups,and the Ct values were 19.22,19.23,19.11 and 18.26,respectively,which indicated that the method had good repeatability.The specific detection of mycobacterium tuberculosis H37Rv,bovine mycobacterium tuberculosis,B.suis S2,B.abortus S19 and BVDV NADL was carried out.The results showed that there was no obvious amplification curve in B.suisS2,BVDV NADL,and template,indicating that the system had good specificity.The correlation coefficient R2 of the obtained kinetic curve was 0.999,showing a good linear correlation.The original concentration of the standard sample was diluted10 times and the minimum detection copy number was 4 Copies/uL,which showed that the method had good sensitivity.The established real-time fluorescence quantitative PCR method was used to validate the clinical samples.The results showed that 100%of the positive tissue samples were detected and 83.33%of the positive deer blood samples were detected.The detection accuracy of the established method was higher than that of the common PCR method.(3)A method for the determination of celazine residues in sika deer antler and deer blood by ultra-high performance liquid chromatography-triple four-stage mass spectrometry was established.The pretreatment,extraction and chromatographic conditions of the samples were optimized,and the precision and accuracy of the method were evaluated.The results showed that the detection limit of serazide in velvet antler and deer blood was 0.1 ng/mL~0.5 ng/mL,the lower limit was 0.2 ng/mL~1.0 ng/mL,and the R~2 value was 0.9968.The standard curve was successfully constructed.The results showed that the method was reproducible,rapid and accurate,and suitable for the detection of veterinary narcotic residues in sika deer antler and deer blood related products.(4)A method for the determination of xylene thiazide residues in sika deer antler and deer blood by triple four-stage gas chromatography-mass spectrometry was established.By optimizing the sample treatment conditions and choosing the chromatographic column,the collected samples could be accurately quantified,and the applicability of the method was investigated.The results showed that the detection limit of xylene thiazide in velvet antler and deer blood was 20 ng/mL and the lower limit was 50 ng/mL.The linear equation was Y=8966X-451812 and R~2 is 0.9919.The standard curve was successfully constructed.The results showed that the method was accurate and efficient,which provided technical support for the quality and safety evaluation of sika deer antler.A real-time fluorescence quantitative PCR system for detection of brucella and mycobacterium tuberculosis in sika deer antler and deer blood was established.Ultra-high performance liquid chromatography-triple quadruple-rod mass spectrometry tandem and triple quadruple-rod gas chromatography-mass spectrometry were used for detection of two veterinary anesthetics.The established method for the determination of pollutants in velvet antler and blood of deer had the characteristics of simple sample pretreatment,strong specificity and high sensitivity.It provides important technical support for the detection of main pathogenic microorganisms and veterinary narcotic residues in sika deer antler and deer blood products,and is of great significance for the improvement and supplement of the food safety assessment system of velvet antler and deer blood related products. |
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