[ Objective] To develop multiplex-PCR for diagnosis of mixed infection caused by Pasteurella multocida （ PM）, Haomophilus parasuis （HPS） and Actinbaci/lus pleuropneumoniae （App）. [ Method ] PCR method was develo...[ Objective] To develop multiplex-PCR for diagnosis of mixed infection caused by Pasteurella multocida （ PM）, Haomophilus parasuis （HPS） and Actinbaci/lus pleuropneumoniae （App）. [ Method ] PCR method was developed to detect single infection caused by PM, HPS or App. The conditions of amplification and primers were optimized, and the multiple-PCR was developed to detect mixed infection of PM, HPS and App. [ Result] A 457-bp band, a 821-bp band and a 342-bp band were simultaneously amplified in the one PCR reaction system. The method had high sensitivity and specificity. [ Conduslon] The multiple-PCR is successfully developed and can be used for differential diagnosis of PM, HPS and App.展开更多
The use of an internal control in a multiplex-PCR assay for sex determination of In Vitro-produced bovine embryos was evaluated in biopsies of groups of 54 fresh and 44 frozen embryos. The internal controls used were ...The use of an internal control in a multiplex-PCR assay for sex determination of In Vitro-produced bovine embryos was evaluated in biopsies of groups of 54 fresh and 44 frozen embryos. The internal controls used were the primers BOV 1 and BOV 2, which amplify a product with 626 base pairs (bp) of bovine mitochondrial DNA ND5 gene. The primers BRY.4aF and BRY.4aR were used for bovine Y chromosome sequence amplification. The specificity of multiplex- PCR reactions realized in biopsies corresponding to about 20% of each fresh embryo (10 male and 10 female) by means of confirming the sexing in the remaining embryo content (~80%) presented 100% specificity. Amplicons of the internal control and Y chromosome were both amplified until dilution corresponding to 6.25% of total extracted DNA from a male embryo. Sex determination was possible in 53 (98.1%) fresh embryos and 40 (90.9%) frozen embryos. The products related to the Y chromosome and mitochondrial DNA were simultaneously amplified in 34 (63%) fresh embryos and 27 (61.4%) frozen embryos, showing a male embryo. The female sex, distinguished by internal control amplification only, was detected in 19 (35.2%) and 13 (29.5%) biopsies, respectively, of fresh and frozen embryos. In one (1.8%) and four (9.1%) biopsies of fresh and frozen embryos, respectively, neither product was amplified, most likely due to the absence of embryonic cells or the presence of embryonic cells going through apoptosis. The multiplex-PCR assay developed in this work showed avoided the limitation of a lack of an internal standard, and was also sensitive, specific, and efficient in reaction failure identification. This technique shows great potential for use on a commercial scale in routine sex determination of In Vitro-produced embryos.展开更多
Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of ...Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of this pathogen exhaustive monitoring of this pathogen is considered of critical importance to public health organizations. The reliable identification method able to distinguish genetic close Pseudomonas species is needed, because these organisms are difficult to differentiate by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect four Pseudomonas species which are frequently detected from the human oral cavities, and to investigate the distribution of these organisms in the living environment using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the rpoD gene of four Pseudomonas species. Swab samples were collected from fifty washstands, and the distribution of Pseudomonas species was investigated using a conventional PCR at genus level and a multiplex PCR at species level. Results: Multiplex PCR method developed in this study was able to distinguish four Pseudomonas species clearly. The genus Pseudomonas was detected from all samples (100%), whereas P. putida, P, aeruginosa, P. stutzeri and P. fluorescens were detected at 44%, 8%, 4% and 2% in fifty swab samples, respectively. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction. It was indicated that washstands were the uninhabitable environment for P. putida, P, aeruginosa, P. stutzeri and P. fluorescens.展开更多
基金funded by the subproject of National Key Technology R&D Program (2006BAD06A01 and 2006BAD06A12)Basic Work of National Science and Technology Special Plan (2008FY210200)
文摘[ Objective] To develop multiplex-PCR for diagnosis of mixed infection caused by Pasteurella multocida （ PM）, Haomophilus parasuis （HPS） and Actinbaci/lus pleuropneumoniae （App）. [ Method ] PCR method was developed to detect single infection caused by PM, HPS or App. The conditions of amplification and primers were optimized, and the multiple-PCR was developed to detect mixed infection of PM, HPS and App. [ Result] A 457-bp band, a 821-bp band and a 342-bp band were simultaneously amplified in the one PCR reaction system. The method had high sensitivity and specificity. [ Conduslon] The multiple-PCR is successfully developed and can be used for differential diagnosis of PM, HPS and App.
文摘The use of an internal control in a multiplex-PCR assay for sex determination of In Vitro-produced bovine embryos was evaluated in biopsies of groups of 54 fresh and 44 frozen embryos. The internal controls used were the primers BOV 1 and BOV 2, which amplify a product with 626 base pairs (bp) of bovine mitochondrial DNA ND5 gene. The primers BRY.4aF and BRY.4aR were used for bovine Y chromosome sequence amplification. The specificity of multiplex- PCR reactions realized in biopsies corresponding to about 20% of each fresh embryo (10 male and 10 female) by means of confirming the sexing in the remaining embryo content (~80%) presented 100% specificity. Amplicons of the internal control and Y chromosome were both amplified until dilution corresponding to 6.25% of total extracted DNA from a male embryo. Sex determination was possible in 53 (98.1%) fresh embryos and 40 (90.9%) frozen embryos. The products related to the Y chromosome and mitochondrial DNA were simultaneously amplified in 34 (63%) fresh embryos and 27 (61.4%) frozen embryos, showing a male embryo. The female sex, distinguished by internal control amplification only, was detected in 19 (35.2%) and 13 (29.5%) biopsies, respectively, of fresh and frozen embryos. In one (1.8%) and four (9.1%) biopsies of fresh and frozen embryos, respectively, neither product was amplified, most likely due to the absence of embryonic cells or the presence of embryonic cells going through apoptosis. The multiplex-PCR assay developed in this work showed avoided the limitation of a lack of an internal standard, and was also sensitive, specific, and efficient in reaction failure identification. This technique shows great potential for use on a commercial scale in routine sex determination of In Vitro-produced embryos.
文摘Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of this pathogen exhaustive monitoring of this pathogen is considered of critical importance to public health organizations. The reliable identification method able to distinguish genetic close Pseudomonas species is needed, because these organisms are difficult to differentiate by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect four Pseudomonas species which are frequently detected from the human oral cavities, and to investigate the distribution of these organisms in the living environment using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the rpoD gene of four Pseudomonas species. Swab samples were collected from fifty washstands, and the distribution of Pseudomonas species was investigated using a conventional PCR at genus level and a multiplex PCR at species level. Results: Multiplex PCR method developed in this study was able to distinguish four Pseudomonas species clearly. The genus Pseudomonas was detected from all samples (100%), whereas P. putida, P, aeruginosa, P. stutzeri and P. fluorescens were detected at 44%, 8%, 4% and 2% in fifty swab samples, respectively. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction. It was indicated that washstands were the uninhabitable environment for P. putida, P, aeruginosa, P. stutzeri and P. fluorescens.
