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At present, the development of viral disease diagnosis technology tends to be specific, sensitive, rapid, economical, safe and simple to operate, can achieve early diagnosis purposes, is suitable for the detection of a large number of samples, and diagnostic techniques tend to be kit-based, PRRS The diagnosis is no exception. At present, the ELISA diagnostic kit for PRRS produced by IDEXX in the United States has been widely used worldwide. The market for diagnostic kits for domestic PRRS is almost monopolized by the US IDEXX company, and this imported ELISA kit is expensive. Farms and laboratories with good conditions can afford it. However, the ELISA kit is coated with a PRRSV whole virus-labeled plate (Steven, eta 1, 2000). Therefore, the manufacturer's required kit material must be incinerated; if there is a mistake in handling or handling, There is a danger of potential poisoning, and the prevalence of PRRS in China may be related to the improper introduction and use of the kit. Therefore, it is necessary to study a PRRS ELISA diagnostic method that is safe, non-disinfecting, and highly sensitive.
The use of recombinant protein as a target antigen to establish a serological diagnostic method can avoid the drawback of sporadic toxicity. Because the N protein of PRRSV is very conservative and highly immunogenic, serology established on the basis of molecular biology The detection methods are based on recombinant N protein expressed in prokaryotic or eukaryotic expression (Dea, eta1, 2000b~Denac, eta1, 1997~Steven, eta [, 200% Meulenberg, et al, 1995b). Dea et al. (2000) directly dissolved in 4 mol/L guanidine hydrochloride by prokaryotic expression of recombinant N protein, which was used in serological methods after dilution with PBS. The EL1SA method for detecting PRRSV antibody was successfully established. 7 to 10 days, PRRSV antibody can be detected from serum; 542 pig serum can be detected simultaneously with 1DEXX company commercial ELISA diagnostic kit, the results of the two tests are completely identical; all 5 epitopes (ORF7) or segmentation It was found that only the intact recombinant N protein (prokaryotic expression) was suitable for the study of PRRS serological diagnosis methods (Dea, et al, 2000b). ORF7 also has been reported to be expressed in the baculovirus expression system, and the established ELISA also showed a good diagnostic effect, but the expression level of the baculovirus expression system is far less than that of the prokaryotic expression, and the required test conditions and costs. High, and the fetal bovine serum added during the cultivation of insect cells is liable to cause a non-specific reaction to the serological reaction, and thus the practical application is limited (Denac, et al, 1997~ Steven, et al, 2000). Domestic researchers have also studied the expression of ORF7 in two systems (Qi Huaji et al., 1999. Wang Yunfeng et al., 2000~ Zhao Wei et al., 2001), but none of the true kit-based methods have emerged and applied to PRRS. Diagnosis. M protein is a conserved protein between European and American PRRSV strains, and has strong immunogenicity. The corresponding antibody can be detected on the 10th day after infection (Meng, etal, 1995b~Loemba, etal , 1996), therefore, the M protein of PRRSV can also be used as a target antigen for PRRS diagnosis, but there is no report on the expression of M protein and the establishment of diagnostic methods at home and abroad. If the N protein is fused to the M protein, the ELISA method established with the recombinant protein MN may be better. This method is worth exploring.
Albina et al (1992) inoculated PAM culture with PRRSV, extracted PRRSV antigen, and established an indirect ELISA for detecting PRRSV antibody for the first time. When the ratio of the OD value of PRRSV antigen well to the OD value of cell antigen pore is greater than 1.5, the sample is judged. Positive. The study found that the specificity of ELISA is the same as that of IPMA, but the sensitivity is higher. Since PAM is a primary cell, it is difficult to obtain sufficient cells for the preparation of PRRSV antigen. Takikawa et al. (1996) used PRRSV to inoculate MARCl45 cells to extract PRRSV antigen, and established an ELISA method for detecting PRRSV antibody. The results showed that ELISA has IPMA and IFA. Better specificity and sensitivity. Since the PRRSV antigen prepared with MAR~145 has a certain degree of non-specific reaction, further improvement is necessary. Cho et al (1996) used Triton-X100 to help dissolve high purity E1 from PRRSV-infected MARC-145 cells. ISA antigen, this high-quality antigen is applied to an indirect ELISA for detecting PRRSV antibodies, supplemented with an effective blocking agent to eliminate non-specific reactions in serum samples, and this ELISA is more sensitive than 1FA, especially for late-stage serum It has high specificity; it has the advantages of rapid, economical, sensitive, suitable for a large number of samples, semi-automatic and automatic display results, and is obviously superior to IPMA and IFA. At present, ELISA has been used as a routine means for PRRS detection and diagnosis.