We evaluated the performance of a new chromogenic medium for detection of Clostridium difficile, chromID C. difficile agar (CDIF; bioMerieux, France), by comparison with BBL C. difficile Selective Agar (CDSA; Becton Dickinson and Company, USA). After heat pre-treatment (80degrees C, 5 min), 185 diarrheal stool samples were inoculated onto the two media types and incubated anaerobically for 24 hr and 48 hr for CDIF and for 48 hr and 72 hr for CDSA. All typical colonies on each medium were examined by Gram staining, and the gram-positive rods confirmed to contain the tpi gene by PCR were identified as C. difficile. C. difficile was recovered from 36 samples by using a combination of the two media. The sensitivity with CDIF 48 hr was highest (100%) and was significantly higher than that with CDIF 24 hr (58.3%; P<0.001), because samples with a low burden of C. difficile tended to require prolonged incubation up to 48 hr (P<0.001). The specificity of CDIF 24 hr and CDIF 48 hr (99.3% and 90.6%, respectively) was significantly higher than that of CDSA 48 hr and CDSA 72 hr (72.5% and 67.1%, respectively; P<0.001). CDIF was effective for detecting C. difficile in heat-pretreated stool specimens, thus reducing unnecessary testing for toxin production in non-C. difficile isolates and turnaround time.
Agar/chemistry ; Bacterial Proteins/genetics ; Bacteriological Techniques/*methods ; Chromogenic Compounds/chemistry/metabolism ; Clostridium difficile/genetics/*isolation & purification ; Culture Media/chemistry ; DNA, Bacterial/analysis/metabolism ; Diarrhea/microbiology/pathology ; Feces/*microbiology ; Humans ; Polymerase Chain Reaction ; Time Factors

OBJECTIVETo determine the antiproliferative activity of Rubus parvifolius L. (RP) extract, its medicinal serum and RP total saponins (RPTS) against K562 cells in vitro and in vivo.

METHODSNude mice models bearing leukemia tumors were treated with different concentrations of RP extract. The size, weight and histopathological change of leukemic tumors were determined. Semi-solid agar culture and methylthiazolyl tetrazolium (MTT) assay were used to determine in vitro the inhibition of colony formation and proliferation of K562 cells respectively by different concentrations of RP medicinal serum and RPTS.

RESULTSRP extract had a tumor inhibition rate of 84.8% when administered to mice at a dose of 1.0 g/day of crude RP root equivalent. Semi-solid agar culture of K562 cells in the presence of 20% (v/v) of RP medicinal serum and 150 mg/L RPTS demonstrated a 50.8% and 100% inhibition of the colony forming unit (CFU)-K562, respectively. The same doses of RP medicinal serum and RPTS showed a proliferation inhibition of 31.4% and 86.3%, respectively against K562 cells in MTT assay.

CONCLUSIONRP extract and RPTS show effective antiproliferative activity against myeloid leukemia cells in vitro and in vivo.

Agar ; Animals ; Cell Proliferation ; drug effects ; Chromatography, High Pressure Liquid ; Humans ; K562 Cells ; Leukemia ; drug therapy ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Plant Extracts ; pharmacology ; therapeutic use ; Rosaceae ; chemistry ; Saponins ; pharmacology ; therapeutic use ; Subcutaneous Tissue ; pathology ; Tumor Stem Cell Assay ; Xenograft Model Antitumor Assays

BACKGROUND: ChromID Clostridium difficile agar (IDCd; bioMerieux SA, France) is a recently developed chromogenic medium for rapid and specific isolation of C. difficile. We compared the performance of IDCd with that of Clostridium difficile Selective Agar (CDSA). METHODS: A total of 530 fresh stool specimens were collected from patients with clinical signs compatible with C. difficile infection, and cultures for C. difficile were performed on IDCd and CDSA. C. difficile colonies were identified by spore staining, odor, use of an ANI identification test kit (bioMerieux SA), and multiplex PCR for tcdA, tcdB, and tpi. RESULTS: The concordance rate between IDCd and CDSA was 90.6% (480/530). The positivity rates on IDCd on days 1 and 2 (55.6% and 85.0%, respectively) were significantly higher than those on CDSA (19.4% and 75.6%, respectively) (P<0.001 for day 1 and P=0.02 for day 2), but the detection rates on IDCd and CDSA on day 3 were not different (89.4% vs. 82.8%, P=0.0914). On day 3, the recovery rates for non-C. difficile isolates on IDCd and CDSA were 30.2% (160/530) and 22.1% (117/530), respectively (P=0.0075). Clostridium spp. other than C. difficile were the most prevalent non-C. difficile isolates on both media. CONCLUSIONS: The culture positivity rates on IDCd and CDSA were not different on day 3 but IDCd may allow for rapid and sensitive detection of C. difficile within 2 days of cultivation.
Agar/*chemistry ; Bacterial Proteins/genetics ; Bacterial Toxins/genetics ; Clostridium difficile/genetics/*isolation & purification ; DNA, Bacterial/analysis ; Enterocolitis, Pseudomembranous/diagnosis/microbiology ; Enterotoxins/genetics ; Feces/*microbiology ; Humans ; Multiplex Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Triose-Phosphate Isomerase/genetics

OBJECTIVETo investigate single-molecule detection and characterization of DNA replication.

METHODSSingle-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication.

RESULTSThe designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis.

CONCLUSIONSThe combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

Biotinylation ; DNA ; chemistry ; DNA Replication ; DNA, Single-Stranded ; chemistry ; DNA-Directed DNA Polymerase ; Electrophoresis, Agar Gel ; Microscopy, Atomic Force ; Nucleic Acid Hybridization ; Streptavidin

OBJECTIVE: To establish miniSTR fluorescent detection system with all detected fragments below 150 bp and to enhance the efficiency of detecting the degraded DNA samples. METHODS: All candidate primers were designed by Primer Premier 5 and screened by FastPCR 6.0. The miniSTR multiplex system was established by these selected loci labeling by four fluorescent dye. The parameters of PCR and primer concentrations were subsequently optimized. The electrophoresis was fulfilled under POP4 on 3100-Avant and the typing data was validated by standard DNA 9947A and 007. Fresh blood samples and difficult degraded DNA samples were tested to evaluate the usefulness of the system. RESULTS: All amplicons in the established miniSTR fluorescent detection system (D12ATA63, D2S1776, D1GATA113, D4S2408, D17S974, D20S482, D3S3053, Amelogenin, D6S474, D9S1122) were less than 150bp. The profile showed a balanced peak height without extra stutter by optimal protocol. Allele frequencies showed no deviations from Hardy-Weinberg equilibrium. The system showed accumulated probability of discrimination 0.999 999 983 and accumulated triplet excluding probability of paternity 0.996 8. It could detect corrupt muscle tissue, low copy number DNA samples and human tissues fixed by 40% formaldehyde solution for 12 days. CONCLUSION The miniSTR fluorescent detection system could be solely used for personal identification of degraded DNA samples or complementally used for paternity tests. And the system could enhance the ability of detecting the trace and degraded DNA.
DNA/chemistry* ; DNA Fingerprinting ; DNA Primers/genetics* ; Electrophoresis, Agar Gel ; Forensic Genetics ; Gene Frequency/genetics* ; Genetic Markers/genetics* ; Genetics, Population ; Humans ; Polymerase Chain Reaction/methods* ; Reference Standards ; Sequence Analysis, DNA/methods*

Tanshinones are the bioactive components of the Chinese medicinal herb Salvia miltiorrhiza, while its biosynthetic pathway remains to be characterized. Rapid identification and characterization of the genes correlated to tanshinones biosynthesis is very important. As one of the intermediates of tanshinones biosynthesis, the ferruginol content is relative low in both root and engineered bacteria. It is urgent to construct an efficient system for conversion of miltiradiene to ferruginol to obtain large amount of ferruginol as the substrates for further identifying other downstream genes involved in tanshinones biosynthesis. In this study, we constructed the whole-cell yeast biocatalysts co-expressing miltiradiene oxidase CYP76AH1 and cytochrome P450 reductases (SmCPR1) from Salvia miltiorrhiza, and then characterized it with RT-PCR. After permeabilization, the yeast whole-cell could catalyze turnover of miltiradiene to ferruginol efficiently through single-step biotransformation with a conversion efficiency up to 69.9%. The yeast whole-cell biocatalyst described here not only provide an efficient platform for producing ferruginol in recombinant yeast but also an alternative strategy for identifying other CYP genes involved in tanshinones biosynthesis.
Biosynthetic Pathways ; Biotransformation ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Diterpenes ; metabolism ; Diterpenes, Abietane ; biosynthesis ; chemistry ; Electrophoresis, Agar Gel ; Gene Amplification ; NADPH-Ferrihemoprotein Reductase ; genetics ; metabolism ; Open Reading Frames ; Plasmids ; Saccharomyces cerevisiae ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry

OBJECTIVE: To compare effects of three different methods for mtDNA extraction from common sarcosaphagous insects including cetyl trimethyl ammonium bromide (CTAB) method, sodium dodecyl sulfate-potassium acetate (SDS-KAc) method and sodium dodecyl sulfate-proteinase K (SDS-PK) method. METHODS: Seventy-two insects from four species [Chrysomya megacephala (Fabricius, 1784), Eusilpha bicolor (Fairmaire, 1896), Paraeutrichopus pecoudi (Mateu, 1954), Vespa velutina (Lepeletier, 1836)] were collected from the corpses of the rabbits in Changsha district. The total DNA of above samples was extracted by CTAB, SDS-Kac and SDS-PK methods. The purity and concentration of DNA were examined by protein-nucleic acid spectrophotometry, and mtDNA were amplified by specific primers and PCR products were detected by agarose gel electrophoresis. Then PCR products were sequenced and subsequently up-loaded to GenBank. RESULTS: mtDNA was successfully extracted with three methods from most of the samples. The SDS-PK method was better in DNA purity compared to other methods and the CTAB method was superior in extracting DNA from old samples, while SDS-KAc method showed no significant difference for extraction effects of different samples. CONCLUSION The most appropriate method should be chosen depending on different situations. SDS-PK method is expected to obtain high-quality DNA, while CTAB method is preferred in extracting obsolete samples. SDS-KAc method is low cost and can be used in various kinds of preliminary experiments.
Animals ; Coleoptera/genetics* ; DNA Primers ; DNA, Mitochondrial/isolation & purification* ; Diptera/genetics* ; Electrophoresis, Agar Gel ; Entomology ; Forensic Medicine/methods* ; Gene Amplification ; Insecta/genetics* ; Polymerase Chain Reaction/methods* ; Quaternary Ammonium Compounds/chemistry* ; Rabbits ; Reproducibility of Results ; Sequence Analysis, DNA ; Sodium Dodecyl Sulfate/chemistry*

BACKGROUND: Accurate and early detection of vancomycin-resistant enterococci (VRE) is critical for controlling nosocomial infection. In this study, we evaluated the usefulness of a selective chromogenic agar medium and of multiplex PCR for detection of VRE, and both these techniques were compared with the conventional culture method for VRE detection. METHODS: We performed the following 3 methods for detecting VRE infection in stool specimens: the routine culture method, culturing in selective chromogenic agar medium (chromID VRE, bioMerieux, France), and multiplex PCR using the Seeplex(R) VRE ACE Detection kit (Seegene Inc., Korea) with additional PCR for vanC genes. RESULTS: We isolated 109 VRE strains from 100 stool specimens by the routine culture method. In chromID VRE, all the isolates showed purple colonies, including Enterococcus gallinarum and E. raffinosus, which were later identified using the Vitek card. All VRE isolates were identified by the multiplex PCR method; 100 were vanA-positive E. faecium, 8 were vanA- and vanC-1-positive E. gallinarum, and 1 was vanA-positive E. raffinosus. CONCLUSIONS: For VRE surveillance, culturing the isolates in chromID VRE after broth enrichment appears to be an accurate, rapid, and easy method for routine screening test. Multiplex PCR is relatively expensive and needs skilled techniques for detecting VRE, but it can be an auxiliary tool for rapid detection of genotype during a VRE outbreak.
Agar/chemistry ; Chromogenic Compounds/*chemistry ; Enterococcus/drug effects/genetics/*isolation & purification ; Enterococcus faecium/genetics/isolation & purification ; Feces/microbiology ; Genotype ; Humans ; Phenotype ; Polymerase Chain Reaction/*methods ; Reagent Kits, Diagnostic ; *Vancomycin Resistance

BACKGROUND: The incidence of Bacillus cereus bacteremia is increasing, but the identification of Bacillus species remains difficult. Brilliance Bacillus cereus agar (BBC agar; Oxoid, UK) is a new CHROMagar medium that allows selective isolation and identification of B. cereus; however, its clinical usefulness is seldom studied. We evaluated the usefulness of BBC agar to identify B. cereus isolates recovered from blood cultures. METHODS: We analyzed a total of 53 blood isolates that showed a Bacillus-like morphology on Gram staining. All isolates were identified by using both the API Coryne (bioMerieux, France) and API 50CH/B (bioMerieux) systems. They were subsequently subcultured on BBC agar, incubated for 24 hr, and then examined for characteristic blue-green colonies. The clinical characteristics of patients whose isolates were identified as B. cereus were assessed. RESULTS: Of the 53 isolates, 18 were identified as B. cereus by API 50CH/B. With the API 50CH/B system used as gold standard, the sensitivity and specificity for the identification of B. cereus were 100% (18/18) and 100% (35/35), respectively, using BBC agar, and 67% (12/18) and 100% (35/35), respectively, using the API Coryne system. Of the 18 patients with B. cereus bacteremia, 15 showed infectious signs, and 3 had more than 2 blood cultures positive for B. cereus on separate days. CONCLUSIONS: Our study shows, for the first time, that BBC agar, with its good agreement and ease of use, is a valuable alternative to the API 50CH/B system for the presumptive identification of B. cereus isolates from blood cultures.
Adolescent ; Adult ; Agar/chemistry ; Aged ; Bacillus cereus/*isolation & purification ; Bacteremia/*diagnosis/microbiology ; Child ; Child, Preschool ; Chromogenic Compounds/*chemistry ; Culture Media ; Female ; Gram-Positive Bacterial Infections/*diagnosis/microbiology ; Humans ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Reagent Kits, Diagnostic ; Sensitivity and Specificity

A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.
Animals ; Colorimetry ; methods ; DNA Primers ; genetics ; Electrophoresis, Agar Gel ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; Influenza A Virus, H3N2 Subtype ; genetics ; Influenza, Human ; diagnosis ; virology ; Naphthalenesulfonates ; chemistry ; Nucleic Acid Amplification Techniques ; methods ; Orthomyxoviridae Infections ; diagnosis ; veterinary ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Swine ; Swine Diseases ; diagnosis ; virology ; Temperature