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W W Cao

Publications and source records attributed to W W Cao.

31 records · Page 2Linked to original sources
PubMed1994

The use of PCR to monitor the population abundance of six human intestinal bacterial species in an in vitro semicontinuous culture system.

Six PCR primer sets complementary to the 16S rDNAs (rRNA genes) were developed and shown to be specific for the following anaerobic bacteria: Clostridium clostridiiforme, C. perfringens, C. leptum, Bacteroides vulgatus, B. distasonis, and B. thetaiotaomicron, respectively. These primers were used for PCR to detect and monitor the bacteria in a semicontinuous culture system designed to mimic intestinal microflora in the human gastrointestinal tract. Except for C. perfringens, the five species of Bacteroides and Clostridia present in the in vitro culture system were detected by the PCR, and the titers varied from 10(-2) to 10(-6) dilutions. The role of azo dye reduction by these bacterial species in the system was examined and discussed.

Bacteria, Anaerobic↗
PubMed1994

A 16S rDNA-based PCR method for rapid and specific detection of Clostridium perfringens in food.

A 16S rDNA-based polymerase chain reaction (PCR) method was developed for the rapid and specific detection of Clostridium perfringens in food. The PCR primers were designed by a GenBank computer search and they are complementary only with the 16S rRNA gene of C. perfringens by sequence alignment. The PCR product is a 279 BP DNA fragment. All C. perfringens strains tested were positive in the PCR assay and all other species tested were negative, including 11 other species of Clostridium and 38 species of other common bacteria. As few as two cells of C. perfringens in pure culture were detectable. High numbers of other bacterial species did not interfere with the detection of C. perfringens. The PCR amplification required only 30 min to complete. The method can be used for detection of C. perfringens in contaminated food. Samples from 100 g of chicken drumsticks which were inoculated with 20, 200 or 2000 cells of C. perfringens were subjected to the PCR assay and all produced positive results.

Animals↗
PubMed1993

A 16S rRNA-based DNA probe and PCR method specific for Listeria ivanovii.

A 16S rRNA-based DNA probe and polymerase chain reaction (PCR) method was developed for identification and rapid detection of Listeria ivanovii. The probe (R-1) is 5'-GTAGTGACGCATGTCATCAC-3' corresponding to positions 185-204 in the L. ivanovii 16S rRNA sequence. DNA hybridization results indicated that R-1 probe only reacted with L. ivanovii, and not with six other species of Listeria or other bacteria tested. The PCR method using R-1 and a reverse primer, R-2, was positive with all eight strains of L. ivanovii tested but was negative with six other species of Listeria, including nine strains of L. monocytogenes, and 20 other taxonomically related bacteria tested. In our PCR method, starting with whole bacterial cells, only 3 h were required for the PCR assay and 1 h for electrophoresis without any additional time for DNA isolation and DNA hybridization. This PCR method detected as few as 4 cells of L. ivanovii in pure cultures and 4-40 cells of L. ivanovii in inoculated and diluted mouse feed, blood, or faeces samples.

Base Sequence↗
PubMed1993

Development of PCR method specific for Marek's disease virus.

A rapid polymerase chain reaction (PCR) assay specific for Marek's Disease Virus (MDV) was developed. This assay was able to detect MDV in inoculated chick kidney cells at dilutions of 10(-5). Negative PCR results were obtained using uninoculated chick cells, Marek's Disease Vaccine (SB), Herpesvirus of Turkeys (HVT) and Fowl Laryngotracheitis Vaccine (LT). Bursae, feathers and kidneys from MDV infected chickens were positive in the PCR assay. The same tissues from normal chickens were negative. This method required only 0.5 h for sample preparatory, 3 h for PCR application and 1 h for electrophoresis. Internal probe hybridization confirmed that the PCR products are from MDV, but this hybridization will not be necessary for future MDV detection.

Animals↗
PubMed1993

Development and evaluation of the polymerase chain reaction method for diagnosis of Mycoplasma gallisepticum infection in chickens.

A polymerase chain reaction (PCR) method specific for Mycoplasma gallisepticum (MG) was evaluated. The PCR method was found to detect as few as two colour changing units (CCU) of MG and did not give false positive reactions with other avian mycoplasmas. In chickens inoculated with either MG or Mycoplasma synoviae (MS), the PCR method was found to closely correlate with MG culture reisolation methods in chicken intranasally inoculated with MG. All chickens inoculated with MS tested negative using the MG PCR method.

Administration, Intranasal↗
PubMed1992

Development of cell surface protein associated gene probe specific for Listeria monocytogenes and detection of the bacteria in food by PCR.

A genomic library of L. monocytogenes was constructed using lambda Zap II-Eco RI and screened with a monoclonal antibody which is specific for a Listeria cell surface protein. Three positive clones each contained a 6.5 kb insert which in E. coli could express the same Listeria protein. The 6.5 kb insert was further digested with Hin dIII and the smaller fragments were subcloned into a plasmid vector (pBluescript) and screened with 32P-labelled genomic DNA from L. monocytogenes or L. innocua. Three clones which were positive with L. monocytogenes and negative with L. innocua were screened and each contained a 2.1 kb insert. The 2.1 kb insert was partly sequenced and some candidate oligomer probes from the sequences were selected and compared with sequences in a Genbank computer search. One such oligomer probe (T7-list) was confirmed to be specific for L. monocytogenes. The probe hybridized with all 28 strains of L. monocytogenes tested, but not with any of six other Listeria species nor 11 other bacteria tested. Using this probe-primer, a PCR method was developed which could detect as few as 2 cfu of L. monocytogenes in pure cultures, and as few as 4-10 cfu of L. monocytogenes when inoculated into foods.

Antibodies, Monoclonal↗
PubMed1992

16S rRNA-based probes and polymerase chain reaction method to detect Listeria monocytogenes cells added to foods.

A rapid polymerase chain reaction (PCR) method was developed for detection of Listeria monocytogenes in foods. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L. monocytogenes, which were previously reported by us to yield a specific nucleic acid probe. Our method included use of a shorter denaturing time, a shorter annealing time, a rapid transition, and an increase in the number of cycles, resulting in good sensitivity. Just 3 h for PCR plus 1 h for electrophoresis was required. Additional time for DNA isolation and DNA hybridization was not needed. This method detected as few as 2 to 20 CFU of L. monocytogenes in pure cultures and as few as 4 to 40 CFU of L. monocytogenes in inoculated (10(8) CFU), diluted food samples. Seven of eight foods, including four poultry products, gave positive results. Only one food sample, soft cheese, gave interference. An internal probe hybridization test was used to confirm that the PCR products were from L. monocytogenes. A specificity test indicated that this PCR method was positive for all 13 strains of L. monocytogenes tested but negative for the other 6 species of Listeria, including 6 strains of L. innocua, and negative for 17 other gram-positive and gram-negative bacteria tested.

Base Sequence↗
PubMed1991

Dried gel hybridization in place of southern hybridization for detection of Listeria monocytogenes DNA fragments.

A simple, sensitive, dried gel DNA hybridization method for detection of Listeria monocytogenes DNA fragments is described. DNA samples were fractionated on an agarose gel. The gel was then denatured in NaOH-NaCl and neutralized in Tris-NaCl. The resulting agarose gel was dried and hybridized with 32P-labelled DNA probe. No transfer to nitrocellulose membranes was used.

Blotting, Southern↗
PubMed1991

Development of a 16S rRNA-based oligomer probe specific for Listeria monocytogenes.

Crude rRNA was isolated from Listeria monocytogenes, L. innocua, and L. ivanovii and sequenced by a reverse transcriptase method. Only two sequence regions were found to differ for L. monocytogenes versus L. innocua or L. ivanovii. Two oligonucleotide probes (RL-1 and RL-2) complementary to these two regions of rRNA of L. monocytogenes were synthesized. The RL-1 probe had one base while the RL-2 probe had two bases which differed for L. monocytogenes versus L. innocua and L. ivanovii. Use of a dried gel hybridization in place of Northern (RNA) hybridization or dot blot hybridization indicated that the RL-2 probe hybridized with all 36 strains of L. monocytogenes tested but not with 6 other Listeria species and 11 other bacteria tested. The RL-2 probe is specific for L. monocytogenes, while the RL-1 probe showed some cross-reactions with other Listeria species. An alkaline phosphatase-labeled RL-2 probe could be used in a dot blot hybridization test and gave good results, but a 32P-labeled RL-2 probe was more sensitive than the nonradioactive probe and the 32P-labeled probe was useable for up to 2 months, even though the 32P was highly degenerated.

Animals↗
PubMed1991

Comparison and modification of rRNA sequencing methods.

In this study, modification of two methods of RNA sequencing resulted in more definitive sequencing bands. In one method of sequencing, the bands of lane A and lane G sometimes were not clear. Modifications of this method by changing the concentrations of ddATP and ddGTP resulted in the bands of lane A and lane G becoming more readable. Although a second sequencing method was found to have clearer bands than the first method, and the bases immediately downstream from the primer binding site could be read by using r-32P-labeled primer, the bands on the top of lane A still were not clear. Modifications of this second method by changing the ddATP/dATP ratio resulted in the bands of lane A becoming much clearer.

Base Sequence↗
PubMed1991

Development of a simplified method for subclass isotyping and screening monoclonal antibodies.

A simplified dot-blot procedure for screening and subclass isotyping of monoclonal antibodies is described in which only 0.5-50 ng of antigen and 1 microliter of antibody are needed to perform the test. The results on the nitrocellulose membrane can be stored indefinitely for future reference. This method is less expensive, uses smaller quantities of antigen and antibody, and is faster than presently used enzyme-linked immunosorbent assay techniques or other dot-blot methods. Monoclonal antibodies against Mycoplasma gallisepticum were screened and isotyped using this technique.

Antibodies, Monoclonal↗
PubMedDate not supplied

Mechanism of the antiproliferative action of leflunomide. A77 1726, the active metabolite of leflunomide, does not block T-cell receptor-mediated signal transduction but its antiproliferative effects are antagonized by pyrimidine nucleosides.

BACKGROUND: Leflunomide, a novel immunosuppressive drug, prolongs experimental graft survival effectively and has been well tolerated in patients with rheumatoid arthritis. A77 1726, the active metabolite of leflunomide, inhibits lymphocyte proliferation in vitro. This study was conducted in Jurkat T cells to investigate the effects of A77 1726 on signal transduction pathways initiated by ligands of the T-cell receptor CD3 complex and to evaluate the effects of A77 1726 on nucleotide biosynthesis. METHODS: Tritiated thymidine incorporation and cell counts quantitated cell proliferation. Spectrofluorescence of Indo/AM dye measured intracellular Ca2+ mobilization. A luciferase assay quantitated interleukin-2 gene promoter activity in stimulated cells transfected with an interleukin-2 promoter-luciferase gene construct. Pyrimidine and purine nucleosides were used to assess antagonism of the antiproliferative activity of A77 1726. RESULTS: (1) A77 1726 dose-dependently inhibited the proliferation of Jurkat T cells (inhibitory concentration of 50% = 6 mumol/L); (2) A77 1726 did not decrease mobilization of intracellular Ca2+ stimulated by phytohemagglutinin or anti-CD3 monoclonal antibody; (3) A77 1726 did not inhibit interleukin-2 gene promoter activity in cells stimulated with ionomycin plus phorbol myristate acetate; (4) inhibition of cell proliferation by A77 1726 was antagonized by addition of uridine, cytidine, or 2(+)-deoxycytidine; (5) addition of uridine 24 hours after treatment with A77 1726 antagonized inhibition of proliferation; (6) A77 1726 was not antagonized by 2'-deoxyuridine, thymidine, adenosine, or guanosine. CONCLUSIONS: (1) A77 1726 inhibited Jurkat T-cell proliferation without inhibiting T-cell receptor-mediated signal transduction events, including tyrosine kinase-dependent intracellular Ca2+ mobilization and activation of the interleukin-2 gene promoter; (2) the antiproliferative effects of A77 1726 on Jurkat T cells are primarily due to interruption of de novo pyrimidine nucleotide biosynthesis. These data provide evidence for a novel in vitro mechanism of the antiproliferative action of this immunosuppressant.

Aniline Compounds↗