Patients With Cervical Cancer Ribonuclease Inhibitor To The Establishment Of The Rapid Detection Of Factor Gene Mutation Analysis And Common Food-borne Pathogens Technology System

Objective: Ribonuclease Inhibitor (RI) is a 50KD acidic protein existing in cytoplasm, which lies in all tissues and organs of the mammals extensively, especially affluent in the placenta of humans. RI can decrease the RNA degradation by RNase A, because RI can inhibit the activity of RNase A for the combination with alkaline RNase. Angiogenin (Ang) is a single strand 14 KD alkaline protein, belonging to RNase A superfamily, and which can induce the formation of newly formed vascular net. The growth of tumor depends on the formation of the newly formed circumambient vascular, they can supply the necessary nourishment for the growth of tumor. There is 35% homology between the amino acid residues of the Ang and RNase A and their similar spatial structure can both combine with RI as ligand. The dissociation constant of the RI-RNase complex was higher than the RI-Ang complex by 60 times, and RI can inhibit the formation of the newly formed vascular effectively. The objective of the paper was to establish an effective method for analysis of gene mutation of Ribonuclease Inhibitor (RNH) in the blood cells of patients with cervical carcinoma, what's more, the RNH of 30 normal persons and 18 patients with cervical carcinoma were detected by means of the method established in the experiment.Method: The RNH lies in the galianconism of the No. 11 chromosomes (11P^15.5), the whole length of the gene is 12310bp, including in 11 extron and 10 intron. The target gene fragments must contain the whole sequence of extron when primers were designed, and several target gene fragments contained two adjacent extron and parts of the adjoining introns, and therewere 11 pairs of such primers. But the longer intron were divided into several fragments eligibly for amplification, and all the target gene fragments were less than 1000 bp, and there were 10 pairs of such primers, so there were 21 pairs of primers in the experiment, and most of the target gene fragments were between 300-700bp. The conditions of Polymerase Chain Reaction (PCR) were optimized by using the genome DNA extracted from the normal persons' blood cells, then the target gene fragments were amplified using the DNA extracted from the normal persons and the patients with cervical carcinoma as mould respectively, the 21 primers were designed as above. β-actin were adopted as the internal standard to verify the results of PCR. Single strand conformation polymorphism (SSCP) was used to the mutation analysis. The amplification products of the normal persons' DNA were used for the optimization of the experiment. The gene mutations were judged by the difference of the electrophorestic mobility shift of the degenerative single strand DNA on the polyacrylamide gel. All the target gene fragments were amplified used the 21 designed primers after optimization.Results: The PCR products were undertaken the electrophoresis on the agarose gel, then the electrophorestic mobility shift of each product(s) of the 21 primers were compared, but there is no significant differences. The most optimized conditions of the SSCP in the experiments were as follows: The ratio of the acrylamide and Methylene-bis-Acrylamide was 49:1, the concentration was 4%, and glycerin was not contained, the thickness of the gel was 1 mm, the voltage was 220v, the temperature was 4℃, buffer solution was 0.5×TBE, the volume of the sample was 5 μ l. The groups of SSCP analysis were same as the agarose gel electrophoresis, the electrophorestic mobility shifts were obtained according to equation of M=Ld/Vt, the fine repeatabilities were checked by the General Linear Model. There were not significant differences in the results of the single strand DNA electrophorestic mobility shift in polyacrylamide gel electrophoresis. The results showed that there was not significant genetic difference between the gene of RI of the normal persons and patients with cervical carcinoma.Objective The aim of our study was to establish the techniques protocols for the rapid detecton of 8 species foodborn pathogen, and we compare the efficiency of different methods for rapid Salmonella detection in whole chicken rinses samples and to optimize the most appropriated method of detection. For the Salmonella typhimurium from the above samples, we also study the relationship among different strains, a molecular epidemiological analysis method — pulsed-field gel electrophoresis (PFGE) for the analysis of DNA restriction fragment length polymorphism (RFLP) in S. tyhpimurium was used.Methods Accroding to the pathogen's high reserve sequence, designing the prime and probe, and the specificity and sensitivity of the rapid protococls were done to evaluate the new methods suitabiltiy. The microbiological culture, the broth culture-PCR, the real-time PCR and VIDAS methods were used for the detection of Salmonella spp. in Whole Chicken Rinses. Each Salmonella typhimurium strain isolated from Whole Chicken Rinses was enriched, the Bacteria Cell Suspension Buffer (CSB) were mixed with melted 1% SeaKem Gold:1% SDS agarose, then dispense part of mixture into appropriate well(s) of reusable plug mold. The total genome DNA in agarose plugs was extracted in turn with lysozyme solution, proteinase K solution and TE buffer. The agarose pulgs were digested with restriction enzyme Xba I, followed by pulsed-field gel electrophoresis (PFGE). Salmonella ser. Braenderup H9812 standards was used. The bands were analyzed with statistics software.use BioNumerics software for database maintenance, tiff image normalization, and analysis and patterncomparisons.Results The rapid detection methods are more sensitive , specific and efficient, the detecting limite for PCR was about 10⁴cfu/mL, 10²cfu/mL for real-time PCR, suitable for Salmonella, Staphylococcus aureus, Shigella, Campylobacter jejuni, O₁₅₇.H₇, Listeria monocytogenes, Vibrio paraheamolyticus and Vibrio cholera. The processing is rapid and simple, will be a routine and practical protocols for detecting and identifying pathogenic microorganisms. Using the above methods for Salmonella, total 56 Whole Chicken Rinses samples were detected, Salmonella were founded in 34 samples by the PCR, 36 positive by Real-time PCR, and 28 by VIDAS. Conclusion The above three detecting method were suitable for the rapid detection of Salmonella in Whole Chicken Rinses. 7 S. tyhpimurium isolated from Whole Chicken Rinses were subtyed using PFGE, the lanes with the Salmonella ser. Braenderup H9812 and 7 S. tyhpimurium had above 15 bands, the similarity between of FJ001 and FJ002 is 95.2%, 96.2% similarity is found between FJ003 and FJ004, the similarity range among total 7 S. tyhpimurium is from 61.0% to 96.2%. Taking 85% similitude as criterion, 7 strains of S. tyhpimurium were divided into 5 PFGE types. The typeability, the high discriminatory power and the stability of PFGE-RFLP make this a valuable method to be used in conjunction with serotyping.