Objective To establish and evaluate a real-time PCR assay to detect Mycoplasma pneumoniae （M.pneumoniae） in clinical specimens. Methods By analysing the whole pl gene sequence of 60 M.pneurnoniae clinical isolates i...Objective To establish and evaluate a real-time PCR assay to detect Mycoplasma pneumoniae （M.pneumoniae） in clinical specimens. Methods By analysing the whole pl gene sequence of 60 M.pneurnoniae clinical isolates in Beijing of China, an optimized real-time PCR assay （MpP1） using pl gene conserved region was designed. The specificity and sensitivity of this assay were evaluated and compared with other two reported assays （RepMpl and Mp181） using 40 positive and 100 negative clinical specimens. Results The detection limit of the new assay was 8.1 fg （about 1-3CFU） M.pneumoniae DNA. The sensitivity of MpP1, RepMpl, and Mp181 assays appeared to be 100%, 100%, and 85%, respectively. Conclusion MpP1 assay is suitable for the detection of M.pneumoniae in Chinese clinical specimens.展开更多
This study was conducted to develop a method for accurate quantification of Mycoplasma hyopneumoniae during vaccine production or experimental research. Primer and probe concentration that gave the highest ΔRn and th...This study was conducted to develop a method for accurate quantification of Mycoplasma hyopneumoniae during vaccine production or experimental research. Primer and probe concentration that gave the highest ΔRn and the lowest Ct were selected to establish the real-time PCR system for the detection of M. hyopneumoniae. Template DNA of M. hyopneumoniae was extracted by boiling under different conditions and detected by real-time PCR to determine the optimal conditions for DNA extraction. Thereafter, intra-and inter-batch reproducibility tests were carried out using a standard plasmid to evaluate the stability of the PCR system. Subsequently, the effect of medium composition on the quantitative detection was evaluated. Finally, the correlation between real-time PCR and CCU method was explored. The optimal primer and probe concentration for real-time PCR were 0.4 and 0.2 μmol/L, respectively. The intra-and inter-batch coefficients of variation(CV) in Ct value of 10~4-10~9 copies/μl standard plasmid were <5%, indicating good reproducibility of the real-time PCR system. Following incubation in a boiling water bath for 10 min, M. hyopneumoniae samples can be used directly as a template in subsequent real-time PCR assays,and good intra-batch and inter-batch reproducibility was observed. The working concentration of KM2 medium should be less than the 1/10 of the concentration of the stock solution to minimize its influence on the quantitative detection. Spearman's correlation analysis revealed that the log of CCU and the log of DNA copy number had a significant positive relationship(r=0.797,P=0.000). Thus, the two methods can be used in combination in the quantitative detection of M. hyopneumoniae. In summary, a rapid, stable and accurate quantitative PCR system for detecting M. hyopneumoniae culture was established in this study, which provides a technical means for accurate quantification of M. hyopneumoniae in vaccine production and laboratory tests.展开更多
Objective: To confirm whether Mycoplasma pneumoniae (MP) are present in reproductive tract of STD patients inChina. Methods: Application of nested PCR (nPCR) and DNAsequencing to test samples of urethral/vaginal swabs...Objective: To confirm whether Mycoplasma pneumoniae (MP) are present in reproductive tract of STD patients inChina. Methods: Application of nested PCR (nPCR) and DNAsequencing to test samples of urethral/vaginal swabs withMP culture confirmation of several nPCR positive patients. Results: 74 of 786 STD patients were positive for MP bynPCR, with a rate of 9.4%. of the 484 male patients, 10.5%were positive, and among the 302 female patients, 7.6%were positive. There was no significant difference betweenthem (P<0.05). of 12 cases of MP positive samples by nPCR,4 cases were first generation culture-positive, and one ofthem passed to the next generation successfully. DNAsequencing was performed on the nPCR product of oneswab sample and one MP culture isolation. The determinedsequence was identical to the typical MP strain. Conclusion: In China, MP are present in reproductivetract of both male and female STD patients.展开更多
[Objective] 303 nasal swabs samples were collected from pigs in farms located in Taizhou city, Jiangsu Province, China from March to December 2012 for the purpose of detecting the presence of Mycoplasma hyopneumoniae,...[Objective] 303 nasal swabs samples were collected from pigs in farms located in Taizhou city, Jiangsu Province, China from March to December 2012 for the purpose of detecting the presence of Mycoplasma hyopneumoniae, the primary agent of Enzootic porcine pneumonia （EPP） in pig herds using the nested PCR and Real time PCR techniques. [Method] Nasal swabs were collected from pigs of different ages＇ i.e. 7, 14, 21, 28, 30 and 35 days old, soaked in sterile 1 xPBS overnight at 4 ℃ and DNA extracted using the TIANamp（R） bacterial DNA kit. The DNA samples underwent amplification under the Mhyo 183 q-PCR and P36 primer Nested PCR systems. [Result] With the Nested PCR assay, 38 （12.5%） out of 303 samples tested positive for the presence of M. hyopneumoniae; with the real time PCR assay 152 （50.2%） tested positive for M. hyopneumoniae. The two assays matched to positively detect Mhyo in 22 （7.3%） samples and again matched in 127 （41.9%） samples negative for Mhyo infection. The pattern of infection in both assays was similar where 7- and 35-day-old piglets in both assays had the highest rates of infection i.e. 15.6% and 18.4% for n-PCR and 53.1% and 56.6% for q-PCR for 7- and 35-day-old piglets respectively. [Conclusion] The results highlight the suitability of both PCR assays in establishing the herd infection status of pigs in field conditions.展开更多
Background Mycoplasma pneumoniae （M. pneumoniae） is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous studies hav...Background Mycoplasma pneumoniae （M. pneumoniae） is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous studies have confirmed that the mutation at position 2063 in domain V of the 23S rRNA is the most prevalent, followed by the mutation at position 2064. Reported molecular detection methods for the identification of these mutations include direct sequencing, restriction fragment length polymorphism analysis, real-time polymerase chain reaction （PCR） with high-resolution melt analysis, and nested PCR-linked with capillary electrophoresis, etc. The most commonly used method for monitoring resistance-conferring mutations in M. pneumoniae is direct DNA sequencing of PCR or nested PCR products. However, these methods are time-consuming, labor-intensive or need expensive equipments. Therefore the development of rapid and sensitive methods is very important for monitoring the resistance globally. Methods In this study, we reported a fast and cost-effective method for detecting 2063 and/or 2064 macrolide resistant mutations from specimens using a modified allele-specific PCR analysis, and all results were compared with the sequencing data. We also analyzed the clinical courses of these samples to confirm the modified allele-specific PCR results. Results Among 97 M. pneumoniae specimens, 88 were found to possess mutations by this method, and all modified allele-specific PCR analysis results were consistent with the sequencing data. The data of the clinical courses of these 97 cases showed that they suffered from severe pneumonia. Erythromycin showed better efficacy on cases from which no macrolide resistance mutation was found on their specimens. However, in some cases from which mutations were detected, erythromycin monotherapy had poor efficacy, and on these patients severe symptoms improved only when azithromycin was added to the treatment. Conclusions The drug-resistant M. pneumoniae is very common in Beijing, China. Our modified allele-specific PCR analysis can identify erythromycin resistant mutations more rapidly from specimens than any other method currently available. Erythromycin is still effective for treating patients infected with the mutation negative M. pneumoniae, but this treatment fails to work on mutant organisms. This method can facilitate clinicians in selecting appropriate therapy within short timescales.展开更多
Mycoplasma hyopneumoniae is an important pathogen causing Mycoplasmal pneumonia of swine, which generally causes secondary infections and mixed infections, thus seriously threats the development of swine industry and ...Mycoplasma hyopneumoniae is an important pathogen causing Mycoplasmal pneumonia of swine, which generally causes secondary infections and mixed infections, thus seriously threats the development of swine industry and resulting in huge economic losses. Using PCR technology has very important significance to the correct diagnosis of Mycoplasmal pneumonia at the early stage. In this paper, specific target genes of Mycoplasma hyopneumoniae, methods for clinical sample collection, key technical factors of DNA sample processing method, and the research progress, main advantages and disadvantages, and application of general PCR technology, multiple PCR technology, nested-PCR technology, real-time fluorescence quantitative PCR technology, gene chip detection technology and loop-mediated isothermal amplification in detection of Mycoplasma hyopneumoniae were summarized, which provided convenience for the effective diagnosis and prevention of Mycoplasmal pneumonia of swine.展开更多
基金supported by the National Key Program for Infectious Diseases of China,No.2008ZX10004-002
文摘Objective To establish and evaluate a real-time PCR assay to detect Mycoplasma pneumoniae （M.pneumoniae） in clinical specimens. Methods By analysing the whole pl gene sequence of 60 M.pneurnoniae clinical isolates in Beijing of China, an optimized real-time PCR assay （MpP1） using pl gene conserved region was designed. The specificity and sensitivity of this assay were evaluated and compared with other two reported assays （RepMpl and Mp181） using 40 positive and 100 negative clinical specimens. Results The detection limit of the new assay was 8.1 fg （about 1-3CFU） M.pneumoniae DNA. The sensitivity of MpP1, RepMpl, and Mp181 assays appeared to be 100%, 100%, and 85%, respectively. Conclusion MpP1 assay is suitable for the detection of M.pneumoniae in Chinese clinical specimens.
基金Supported by National Key Research and Development Project of China(2017YFD0501604)National Natural Science Foundation of China(31400164)
文摘This study was conducted to develop a method for accurate quantification of Mycoplasma hyopneumoniae during vaccine production or experimental research. Primer and probe concentration that gave the highest ΔRn and the lowest Ct were selected to establish the real-time PCR system for the detection of M. hyopneumoniae. Template DNA of M. hyopneumoniae was extracted by boiling under different conditions and detected by real-time PCR to determine the optimal conditions for DNA extraction. Thereafter, intra-and inter-batch reproducibility tests were carried out using a standard plasmid to evaluate the stability of the PCR system. Subsequently, the effect of medium composition on the quantitative detection was evaluated. Finally, the correlation between real-time PCR and CCU method was explored. The optimal primer and probe concentration for real-time PCR were 0.4 and 0.2 μmol/L, respectively. The intra-and inter-batch coefficients of variation(CV) in Ct value of 10~4-10~9 copies/μl standard plasmid were <5%, indicating good reproducibility of the real-time PCR system. Following incubation in a boiling water bath for 10 min, M. hyopneumoniae samples can be used directly as a template in subsequent real-time PCR assays,and good intra-batch and inter-batch reproducibility was observed. The working concentration of KM2 medium should be less than the 1/10 of the concentration of the stock solution to minimize its influence on the quantitative detection. Spearman's correlation analysis revealed that the log of CCU and the log of DNA copy number had a significant positive relationship(r=0.797,P=0.000). Thus, the two methods can be used in combination in the quantitative detection of M. hyopneumoniae. In summary, a rapid, stable and accurate quantitative PCR system for detecting M. hyopneumoniae culture was established in this study, which provides a technical means for accurate quantification of M. hyopneumoniae in vaccine production and laboratory tests.
文摘Objective: To confirm whether Mycoplasma pneumoniae (MP) are present in reproductive tract of STD patients inChina. Methods: Application of nested PCR (nPCR) and DNAsequencing to test samples of urethral/vaginal swabs withMP culture confirmation of several nPCR positive patients. Results: 74 of 786 STD patients were positive for MP bynPCR, with a rate of 9.4%. of the 484 male patients, 10.5%were positive, and among the 302 female patients, 7.6%were positive. There was no significant difference betweenthem (P<0.05). of 12 cases of MP positive samples by nPCR,4 cases were first generation culture-positive, and one ofthem passed to the next generation successfully. DNAsequencing was performed on the nPCR product of oneswab sample and one MP culture isolation. The determinedsequence was identical to the typical MP strain. Conclusion: In China, MP are present in reproductivetract of both male and female STD patients.
基金Supported by the Agricultural Science Independent Innovation Foundation of Jiangsu Province[CX(12)1001]~~
文摘[Objective] 303 nasal swabs samples were collected from pigs in farms located in Taizhou city, Jiangsu Province, China from March to December 2012 for the purpose of detecting the presence of Mycoplasma hyopneumoniae, the primary agent of Enzootic porcine pneumonia （EPP） in pig herds using the nested PCR and Real time PCR techniques. [Method] Nasal swabs were collected from pigs of different ages＇ i.e. 7, 14, 21, 28, 30 and 35 days old, soaked in sterile 1 xPBS overnight at 4 ℃ and DNA extracted using the TIANamp（R） bacterial DNA kit. The DNA samples underwent amplification under the Mhyo 183 q-PCR and P36 primer Nested PCR systems. [Result] With the Nested PCR assay, 38 （12.5%） out of 303 samples tested positive for the presence of M. hyopneumoniae; with the real time PCR assay 152 （50.2%） tested positive for M. hyopneumoniae. The two assays matched to positively detect Mhyo in 22 （7.3%） samples and again matched in 127 （41.9%） samples negative for Mhyo infection. The pattern of infection in both assays was similar where 7- and 35-day-old piglets in both assays had the highest rates of infection i.e. 15.6% and 18.4% for n-PCR and 53.1% and 56.6% for q-PCR for 7- and 35-day-old piglets respectively. [Conclusion] The results highlight the suitability of both PCR assays in establishing the herd infection status of pigs in field conditions.
基金Beijing Natural Science Foundation，Beijing City Talent Training Project Fund
文摘Background Mycoplasma pneumoniae （M. pneumoniae） is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous studies have confirmed that the mutation at position 2063 in domain V of the 23S rRNA is the most prevalent, followed by the mutation at position 2064. Reported molecular detection methods for the identification of these mutations include direct sequencing, restriction fragment length polymorphism analysis, real-time polymerase chain reaction （PCR） with high-resolution melt analysis, and nested PCR-linked with capillary electrophoresis, etc. The most commonly used method for monitoring resistance-conferring mutations in M. pneumoniae is direct DNA sequencing of PCR or nested PCR products. However, these methods are time-consuming, labor-intensive or need expensive equipments. Therefore the development of rapid and sensitive methods is very important for monitoring the resistance globally. Methods In this study, we reported a fast and cost-effective method for detecting 2063 and/or 2064 macrolide resistant mutations from specimens using a modified allele-specific PCR analysis, and all results were compared with the sequencing data. We also analyzed the clinical courses of these samples to confirm the modified allele-specific PCR results. Results Among 97 M. pneumoniae specimens, 88 were found to possess mutations by this method, and all modified allele-specific PCR analysis results were consistent with the sequencing data. The data of the clinical courses of these 97 cases showed that they suffered from severe pneumonia. Erythromycin showed better efficacy on cases from which no macrolide resistance mutation was found on their specimens. However, in some cases from which mutations were detected, erythromycin monotherapy had poor efficacy, and on these patients severe symptoms improved only when azithromycin was added to the treatment. Conclusions The drug-resistant M. pneumoniae is very common in Beijing, China. Our modified allele-specific PCR analysis can identify erythromycin resistant mutations more rapidly from specimens than any other method currently available. Erythromycin is still effective for treating patients infected with the mutation negative M. pneumoniae, but this treatment fails to work on mutant organisms. This method can facilitate clinicians in selecting appropriate therapy within short timescales.
基金Supported by National Natural Science Foundation of China(31100135)Agricultural Independent Innovation Fund of Jiangsu Province[CX(11)4038]~~
文摘Mycoplasma hyopneumoniae is an important pathogen causing Mycoplasmal pneumonia of swine, which generally causes secondary infections and mixed infections, thus seriously threats the development of swine industry and resulting in huge economic losses. Using PCR technology has very important significance to the correct diagnosis of Mycoplasmal pneumonia at the early stage. In this paper, specific target genes of Mycoplasma hyopneumoniae, methods for clinical sample collection, key technical factors of DNA sample processing method, and the research progress, main advantages and disadvantages, and application of general PCR technology, multiple PCR technology, nested-PCR technology, real-time fluorescence quantitative PCR technology, gene chip detection technology and loop-mediated isothermal amplification in detection of Mycoplasma hyopneumoniae were summarized, which provided convenience for the effective diagnosis and prevention of Mycoplasmal pneumonia of swine.
