Specific Detection of Toxigenic Vibrio cholerae Based on in situ PCR in Combination With Flow Cytometry

Objective To develop an in situ PCR in combination with flow cytometry （ISPCR-FCM） for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene （ctxAB gene）. Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low （10^5 cells/mL）, while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.

IN SITU PCR AND IMMUNOHISTOCHEMICAL STUDIES ON p16 GENE IN PITUITARY ADENOMAS

Objective: To examine the occurrence of p16 gene deletion and to analyze p16 expression on paraffin-embedded human pituitary adenoma specimens. Efforts were made to optimize the technical conditions forin situ PCR. Methods:In situ PCR techniques and inimuno-histochemistry were used. Results: Immunohistochemically, p16-positive tumor cells were observed in all cases with various proportions. The majority of the stromal cells and part of tumor cells was devoid of p16 immunostaining, but signal ofin situ PCR for p16 gene, exon 2, was displayed in these cells. Conclusion: The results implied that p16 gene might not be deleted in these pituitary adenomas. It also indicated thatin situ PCR, both direct and indirect methods, proved feasible and informative to the aim of DNA detection. It is critical to overcome non-specific amplification in directin situ PCR by means of higher annealing temperature, fewer cycle, lower magnesium concentration and stringent washing. A target DNA-deleted sample as the negative control is extremely necessary. For the indirect method, the way to improve the sensitivity is to loosen the conditions for amplification and washing, so that more amplification products are subject to hybridization, and signal detection is facilitated.

Studies on in situ PCR Detected by the BrdU Antibody Technique

Using the BrdU antibody technique followed by an immuno-chemical staining(BAT),the amplification o f DNA fragments specific to human Y chromosome on cell specimen slides was efficiently detected. Whether direct BrdU incorporation into PCR products or in situ hybridization with PCR products on slides, the amplified targetDNA fragments of specimen were visualized by BAT under the microscope. The availability of BAT and differencesin the sensitivity and efficiency between BAT and dig--if-dUTP labeling in cell in situ PCR were disCussed.

A Study of the Distribution of Apple stem pitting virus in Tissues of Pear Tree Using In Situ Hybridization and In Situ RT-PCR

To understand the distribution of Apple stem pitting virus （ASPV） in tissues of pear tree and provide directly theorical and technical support for shoot tips detoxication, leaves and shoot tips of Korla pear were used as the materials, cDNA probe for ASPV was synthesized through RT-PCR reaction system using the unradioactive digoxigenin-labeled probe, and the specificity and sensitivity of probe were verified by blot hybridization method. Paraffin slice for in situ PCR and in situ hybridization was made and the location and distribution of ASPV RNA were detected in paraffin slices using in situ reverse transcription polymerase chain reaction, and the important factors which influenced the experiment results were optimized. ASPV mainly distributed in palisade tissue of mesophyll cells, external cortex of the tip, and the corresponding newborn vascular bundles. 20 min was the suitable digestive time for proteinase K. For the better amplication, RT reaction system should be above 0.2 U μL^-1 and 0.4 mmol L^-1 for RNasin and dNTPs respectively, 0.1-1.3 U μ L^-1 SuperScript Ⅱ, 0.6- 0.8 μmol L^-1 primer concentration, and above 0.5 U 100 μL^-1 LA Taq DNA polymerase. The suitable annealing temperature in PCR reaction was 60℃ with 35 cycles. The apical meristem of 0.25 mm was the region of virus-free.

In situ RT-PCR detection of inducible nitric oxide synthetase gene expression in lung during endotoxemia in rabbits

To detect the location of inducible nitric oxide synthetase (iNOS) protein and mRNA in lung during endotoxemia in rabbits Methods Northern blotting was performed before, 1 hour and 5 hours after the intravenous administration of lipopolysaccharide (LPS) in rabbits Immuno^histochemical analysis (IA), in situ hybridization and in situ reverse transcription polymerase chain reaction (in situ RT PCR) were also performed in lung sections Results iNOS mRNA expression was found using Northern blotting in lung 5 hours after LPS injection, while it was not found in control The positive stain was found only in macrophages in lung 5 hours after LPS injection by standard hybridization and IA; while by in situ RT PCR, the amplification products were found in macrophages, airway epithelial cells, vascular endothelial cells, smooth muscle cells and leukocytes, in addition to macrophages distributed abundantly throughout the lung The signal was absent in control or samples Conclusions Using an in situ RT PCR technique, iNOS expression was not only observed in macrophages but also in many other kinds of cells in lung during endotoxemia in rabbits This suggests that in situ RT PCR is much more sensitive than in situ hybridization, and can be used to examine genes with low expression

Detection of HBV DNA in Extrahepatic Tissue with PCR Comparison with Immunohistochemistry and in Situ Hybridization

Formalin-fixed paraffin-embedded tissue,including gallbladder,kidney,spleen,adrenal gland,heart,testicle,pancreas,and liver from 18 autopsied cases with HBV infection were studied with nested polymerase chain reaction

Effect of Saccharomyces cerevisiae on alfalfa nutrient degradation characteristics and rumen microbial populations of steers fed diets with different concentrate-to-forage ratios

Live yeast（Saccharomyces cerevisiae） constitutes an effective additive for animal production;its probiotic effect may be related to the concentrate-to-forage ratio（CTFR）.The objective of this study was to assess the effects of S.cerevisiae（SC） on fiber degradation and rumen microbial populations in steers fed diets with different levels of dietary concentrate.Ten Simmental × Local crossbred steers（450 ± 50 kg BW） were assigned to a control group or an SC group.Both groups were fed the same basal diet but the SC group received SC supplementation（8 × 10^9 cfu/h/d through the ruminal fistula）following a two-period crossover design.Each period consisted of four phases,each of which lasted 17 d：10 d for dietary adaptation,6 d for degradation study,and 1 d for rumen sample collection.From the 1^（st） to the 4^（th） phase,steers were fed in a stepwise fashion with increasing CTFRs,i.e.,30：70,50：50,70：30,and 90：10.The kinetics of dry matter and fiber degradation of alfalfa pellets were evaluated;the rumen microbial populations were detected using real-time PCR.The results revealed no significant（P〉 0.05） interactions between dietary CTFR and SC for most parameters.Dietary CTFR had a significant effect（P〈 0.01） on degradation characteristics of alfalfa pellets and the copies of rumen microorganism;the increasing concentrate level resulted in linear,quadratic or cubic variation trend for these parameters.SC supplementation significantly（P〈 0.05） affected dry matter（DM） and neutral detergent fiber（NDF）degradation rates（c_（DM）,c_（NDF）） and NDF effective degradability（ED_（NDF））.Compared with the control group,there was an increasing trend of rumen fungi and protozoa in SC group（P 〈 0.1）;copies of total bacteria in SC group were significantly higher（P〈 0.05）.Additionally,percentage of Ruminobacter amylophilus was significantly lower（P〈 0.05）but percentage of Selenomonas ruminantium was significantly higher（P〈 0.05） in the SC group.In a word,dietary CTFR had a significant effect on degradation characteristics of forage and rumen microbial population.S.cerevisiae had positive effects on DM and NDF degradation rate or effective degradability of forage;S.cerevisiae increased rumen total bacteria,fungi,protozoa,and lactate-utilizing bacteria but reduced starch-degrading and lactate-producing bacteria.