Objective To establish a multiplex polymerase chain reaction （M-PCR） assay for simultaneous detection of pathogens causing genital ulcer disease （GUD）. Mothods Based on the gene-specific region of the following p...Objective To establish a multiplex polymerase chain reaction （M-PCR） assay for simultaneous detection of pathogens causing genital ulcer disease （GUD）. Mothods Based on the gene-specific region of the following pathogens： Chlamydia trachomatis omp l/ompb, herpes simplex virus （HSV） DNA polymerase, Treponema pollidum tpp47, Haemophilus ducreyi 16s rRNA, four sets of primers were designed and an M-PCR assay was developed to detect four pathogens in one test. The assay was evaluated with diagnostic result of golden standard for each pathogen.Results Of the 51 clinical samples, M-PCR showed slightly higher positive rate （47.1%） of HSV than cell culture （23.6%）. Meanwhile, the positive rate of T. pallidum detected by M-PCR and dark-field microscopy was 19.6% （10/51） and 15.7% （8/51）, respectively. Only one sample was positive for H. ducreyi and no sample was positive for C. trachomatis detected by both M-PCR assay and culture. Conclusion This primary study indicated that M-PCR assay can simultaneously and rapidly detect the four etiologic pathogens causing GUD.展开更多
This study differentiated pseudocondyloma of vulva from condyloma acuminata using dot blot hybridization and polymerase chain reaction (PCR). A total of 27 cases of pseudocondyloma of vulva and 65 cases of condyloma a...This study differentiated pseudocondyloma of vulva from condyloma acuminata using dot blot hybridization and polymerase chain reaction (PCR). A total of 27 cases of pseudocondyloma of vulva and 65 cases of condyloma acuminata were selected for the study. The genital lesions were examined clinically and were biopsied. Each biopsy was subjected to histological examination and HPV DNA analysis by dot blot hybridization and PCR. Dot blot analysis detected HPV DNA in 19(82. 6%) out of 23 cases of condyloma acuminata and 2(25% ) out of 8 cases pseudocondyloma of vulvae (P<0. 05). PCR detected HPV DNA in 51 (92. 7%) out of 55 cases of condyloma acuminata , compared with none in 23 cases of pseudocondyloma (P<0.001 ). HPV DNA was present in the majority of condyloma acuminata specimens. HPV 6 and 11 were the predominant types. Peudocondyloma is probably not associated with HPV. PCR was the most sensitive and useful technique for HPV DNA detection.展开更多
Polymerase chain reaction (PCR) was used to detect the presence of Barrelia burgdoferi DNA in biological samples from patients with sarcoidosis. The target DNA sequence was of chromosomal origin. The amplified DNA seq...Polymerase chain reaction (PCR) was used to detect the presence of Barrelia burgdoferi DNA in biological samples from patients with sarcoidosis. The target DNA sequence was of chromosomal origin. The amplified DNA sequence was analyzed by agarose gel electrophoresis, PAGE with silver staining, and the identity of amplified DNA was confirmed by restriction enzyme cleavage and DNA-DNA hybridization with a 32P-labelled probe. The assay was sensitive to fewer than two copies of B. burgdorferi genome, even in the presence of a 104-fold excess of human eukaryotic DNA , and was also specific to different B. burgdorferi strains tested. Sera serologically positive to B. burgdcrferi (n=26), bronchoalveolar lavage fluid and supernatant of BALF (n=26) and peripheral blood (n=9) from sarcoidosis patients were tested. The positive rate was low (4/26, 2/26. and 0/9, respectively). It was considered that DNA from B. burgdor ferimay be identified in a minority of patients with sarcoidosis, and it may play a pathogenetic role in such cases. More studies need to be done before advancing the hypothesis of an etiologic role of B. burgdorferi in sarcoidosis.展开更多
One oligonueleotide probe,HIV env 101-1,and two oligonucleotide primers,HIV env125-1 and HIV env 125-2,were designed with the aid of a computer and synthesized by a DNAsynthesizer.The env 125 peptide gene was amplifie...One oligonueleotide probe,HIV env 101-1,and two oligonucleotide primers,HIV env125-1 and HIV env 125-2,were designed with the aid of a computer and synthesized by a DNAsynthesizer.The env 125 peptide gene was amplified by polymerase chain reaetion(PCR).Beingidentified by electrophoresis and Southern blotting,the PCR product was cloned into plasmidpUC 19.The recombinant pENV 125,identified with X-gal selection and restriction mapping, wassequenced.The results showed that the cloned env 125 peptide gene contained the inserted EcoRⅠ site and ATG at the 5’end,Hind Ⅲ site and TAG at the 3’end.The sequence and readingframe were proved to be correct.展开更多
The sensitivity of PCR was determined for detection of HBV DNA in paraffin-embed-ded liver tissue with different methods for sample preparation.Of 8 cases of HBeAg-positiveHBV infection,HBV DNA was detected in 6 by ex...The sensitivity of PCR was determined for detection of HBV DNA in paraffin-embed-ded liver tissue with different methods for sample preparation.Of 8 cases of HBeAg-positiveHBV infection,HBV DNA was detected in 6 by extracting DNA from both frozen and dewaxedsamples,but in none by direct reaction.Of 12 cases subjected to Southern blot hybridization,HBV DNA was detected in 7 by this technique,in 10 by PCR with both methods of DNA extrac-tion and in 3 by direct PCR.The results showed that PCR was sensitive and was comparablewith blot hybridization in detecting intrahepatic HBV DNA.In comparison between differentmethods of sample preparation,the viral detection rate from the dewaxed samples was near thatfrom the frozen ones,while by the direct reaction HBV DNA could be detected only in a fewsamples with high level of infection.展开更多
To study the diagnosis of hepatitis C viremia,17 patients(including 14 in acute phase)with post-transfusion non-A,non-B hepatitis(NANBH)were investigated by nested polymerasechain reaction after reverse transcription ...To study the diagnosis of hepatitis C viremia,17 patients(including 14 in acute phase)with post-transfusion non-A,non-B hepatitis(NANBH)were investigated by nested polymerasechain reaction after reverse transcription with 3 sets of primers complemented to different regions ofthe viral sequence.Hepatitis C virus(HCV)RNA was detected in 6(35%)cases with reverse tran-script polymerase chain reaction(RT-PCR)using the primers according to the Japanese clones;in13(76%)using the primers according to the non-coding region which is highly conserved in theviral sequence,and in 16(94%)using both or either one of these two sets of primers.However,no HCV RNA was detected with the primers according to the Chiron prototype.The nucleotide se-quence of HCV is quite variable,and the virus level in blood is very low,therefore,in order to getprecise detection,it is suggested to do nested RT-PCR with several sets of primers complementaryto different conserved regions.展开更多
On the basis of the characteristics of Y-chromosome sequence which consists of Y specific repeat DNA family(DYZI) of 800￣5 000 copies,a pair of primers Y3,Y4 is designed to amplify the specific DNA segment of 446 bp ...On the basis of the characteristics of Y-chromosome sequence which consists of Y specific repeat DNA family(DYZI) of 800￣5 000 copies,a pair of primers Y3,Y4 is designed to amplify the specific DNA segment of 446 bp by the polymerase chain reaction(PCR),so as to detect the presence of Y chromosome in male fetal cell in maternal peripheral blood for the purpose of prenatal determination of fetal sex.The authors made use of PCR amplification of crude DNA from maternal peripheral blood in early, mid,and late pregnancies to determine the fetal sex.Comparison of the results with those of PCR of their corresponding chorionic villi and amniotic fluid,and with the sex of the aborted fetus or the new born showed coincident rates of 93%,100%,and 87. 5% in early, mid,and late pregnancies respectively.The procedure offers a new alternative way for non-invasive prenatal diagnosis,展开更多
in the present study,9 exon-containing DNA segments of dystrophin gene with 9 sets of oligonucleotide primers by two-step multiplex polymerase chain reaction (mPCR) were amplified. Subsequently,gene analysis was perfo...in the present study,9 exon-containing DNA segments of dystrophin gene with 9 sets of oligonucleotide primers by two-step multiplex polymerase chain reaction (mPCR) were amplified. Subsequently,gene analysis was performed in 36 cases of Duchenne mascular dystroply (DMD) and 4 cases of Becker muscular dystrophy(BMD). The findings showed that 17 cases of deletion were detected by using the first 5 sets of primers with a relatively high incidence of deletion detection and 2 more cases of deletion were detected by using the remaining 4 sets of primers. The total deletion rate detected by mPCR with 9 cases of primers was 47. 5% of the patients examined,suggesting that about 79. 1% of the patients with gene deletion could be detected. Thus,as a preliminary screening, the two-step mPCR can be used in the gene diagnosis of DMD/BMD. The method is not only simple, convenient and rapid,but also free from radiosotope trouble.展开更多
The polymerase chain reaction (PCR) was used for the detection of toxigenic Clostridium difficile (Cd). A primer pair derived from non-repeating sequences of the toxin A gene were used to amplify a 306 bp DNA fragment...The polymerase chain reaction (PCR) was used for the detection of toxigenic Clostridium difficile (Cd). A primer pair derived from non-repeating sequences of the toxin A gene were used to amplify a 306 bp DNA fragment. Amplified products were visualized by polyacrylamide gel electrophoresis followed by ethidium bromide staining. All 19 strains of toxigenic Cd generated single specific amplified DNA. In contrast, none of the 8 strains of non-toxigenic Cd, 2 strains of C. sordelli and 2 strains of E. coli gave positive results. After the detected DNA of toxigenic Cd was diluted to 0.5ng, the polymerase chain reaction assays were still positive. The results demonstrate that polymerase chain reaction is a simple, rapid, specific and sensitive method for the detection of toxigenic Cd and could be used for the direct detection of Cd in feces samples.展开更多
The cDNA fragment of hepatitis C virus (HCV)was obtained by polymerase chain reaction(PCR) using non-coding region primers. Then this fragment was used as a template to produce single strand(SS) HCV cDNA with asymmetr...The cDNA fragment of hepatitis C virus (HCV)was obtained by polymerase chain reaction(PCR) using non-coding region primers. Then this fragment was used as a template to produce single strand(SS) HCV cDNA with asymmetric PCR. This sscDNA can be used as template for sequencing directly. The result is in line with that reported by Takamizawa.This method is both simple and convenient,and can be used also in other molecular virological studies.展开更多
To assess the presence of HHV-6-Specific DNA in human salivary glands, eighteen specimens of salivary gland tissue were investigated using the polymerase chain reaction. Eight of nine parotid glands, five of seven sub...To assess the presence of HHV-6-Specific DNA in human salivary glands, eighteen specimens of salivary gland tissue were investigated using the polymerase chain reaction. Eight of nine parotid glands, five of seven submandibular glands and one of two sublingual glands were found to have amplification of the HHV-6-specific sequence. The findings suggest that salivary gland tissue is one of the potential sites for HHV-6 persistence following primary infection and that saliva is a vehicle for transmission of the virus.展开更多
In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samp...In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samples of 113 cases of HCC and 69 controls (including 30 cases of liver cirrhosis, 9 cases of metastatic liver cancer and 30 normal subjects). 20/43 (46. 5% ) cases of HCC and 2/30 (6. 7% ) cases of liver cirrhosis are positive and the cases of nletastatic liver cancer and normal controls were negative for human AFP(hAFP) rnRNA. The presence of hAFP mRNA in the peripheral blood seems to be correlated with intrahepatic and distant nletastasls of HCC and portal vein thrombosis. It is concluded that the presence of hAFP mRNA in the peripheral hloocl is an indicator of circulating HCC cells and can be used to diagnose the rnetastasisof HCC through henlatogenous route and RT-PCR amplification of hAFP mRNA is a sensitive and specificprocedure for detecting circulating cells of HCC.展开更多
A nested polymerase chain reaction(N-PCR)for the spegific detection of Helicobacterpylori(H.pylori)was developed with two primer pairs(nested primers)derived from ureasegene A of H.pylori.The N-PCR was used to detect ...A nested polymerase chain reaction(N-PCR)for the spegific detection of Helicobacterpylori(H.pylori)was developed with two primer pairs(nested primers)derived from ureasegene A of H.pylori.The N-PCR was used to detect 21 different samples of H.pylori including20 clinical isolates and 1 reference strain NCTC 14126,but it was negative for other bacterialspecies,showing the N-PCR assay to be 100% specific.Tenfold serial dilution experiments re-vealed the detection of as little as 0.1 fg of H.pylori DNA by N-PCR.To evaluate the PCR as-say for clinical samples,gastric biopsies were tested with N-PCR,and the results were comparedwith those of culture,urease test and histologic examination(reference standard,RS).In 30biopsy specimens,H.pylori DNA sequences were detected by PCR in all of 20(100%)positivetissue and none of the 10 negative tissues.PCR is a specific and sensitive method that can detectthe presence of H.pylori without the need for culture and would have significant importance di-agnostically and epidemiologically.展开更多
目的根据DNA旋转酶B亚基(gyrB)基因设计特异性的引物探针,建立一种能够快速准确鉴定阪崎克罗诺杆菌的实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)方法。方法寻找并在美国国家生物技术信息中心(National Center for Biot...目的根据DNA旋转酶B亚基(gyrB)基因设计特异性的引物探针,建立一种能够快速准确鉴定阪崎克罗诺杆菌的实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)方法。方法寻找并在美国国家生物技术信息中心(National Center for Biotechnology Information,NCBI)中下载目标基因序列,使用DNAMAN进行序列比对,Primer Express软件设计引物探针。通过特异性实验、绝对灵敏度、相对灵敏性实验、抗干扰实验对所建立方法进行方法验证。选择本实验室保存的36种40株食品中常见致病菌的标准菌株进行特异性验证。结果多维度特异性验证实验结果显示该方法能够特异性地检测出阪崎克罗诺杆菌,对亲缘关系较近的其他克罗诺杆菌及食品中较为常见的致病菌均无非特异性扩增。DNA检测灵敏度可以达到0.0100 ng/μL,相对灵敏度可以达到10^(3) CFU/mL。抗干扰实验结果显示,将干扰菌和干扰菌DNA分别与阪崎克罗诺杆菌和阪崎克罗诺杆菌DNA进行混合检测,对检测结果无显著影响,说明该方法抗干扰能力良好。结论本研究所设计的引物探针在实时荧光PCR方法下对食品样品中阪崎克罗诺杆菌的检测具有特异、快速、敏感和抗干扰的特点,可为以后食品中阪崎克罗诺杆菌的检测提供技术支撑。展开更多
文摘Objective To establish a multiplex polymerase chain reaction （M-PCR） assay for simultaneous detection of pathogens causing genital ulcer disease （GUD）. Mothods Based on the gene-specific region of the following pathogens： Chlamydia trachomatis omp l/ompb, herpes simplex virus （HSV） DNA polymerase, Treponema pollidum tpp47, Haemophilus ducreyi 16s rRNA, four sets of primers were designed and an M-PCR assay was developed to detect four pathogens in one test. The assay was evaluated with diagnostic result of golden standard for each pathogen.Results Of the 51 clinical samples, M-PCR showed slightly higher positive rate （47.1%） of HSV than cell culture （23.6%）. Meanwhile, the positive rate of T. pallidum detected by M-PCR and dark-field microscopy was 19.6% （10/51） and 15.7% （8/51）, respectively. Only one sample was positive for H. ducreyi and no sample was positive for C. trachomatis detected by both M-PCR assay and culture. Conclusion This primary study indicated that M-PCR assay can simultaneously and rapidly detect the four etiologic pathogens causing GUD.
文摘This study differentiated pseudocondyloma of vulva from condyloma acuminata using dot blot hybridization and polymerase chain reaction (PCR). A total of 27 cases of pseudocondyloma of vulva and 65 cases of condyloma acuminata were selected for the study. The genital lesions were examined clinically and were biopsied. Each biopsy was subjected to histological examination and HPV DNA analysis by dot blot hybridization and PCR. Dot blot analysis detected HPV DNA in 19(82. 6%) out of 23 cases of condyloma acuminata and 2(25% ) out of 8 cases pseudocondyloma of vulvae (P<0. 05). PCR detected HPV DNA in 51 (92. 7%) out of 55 cases of condyloma acuminata , compared with none in 23 cases of pseudocondyloma (P<0.001 ). HPV DNA was present in the majority of condyloma acuminata specimens. HPV 6 and 11 were the predominant types. Peudocondyloma is probably not associated with HPV. PCR was the most sensitive and useful technique for HPV DNA detection.
文摘Polymerase chain reaction (PCR) was used to detect the presence of Barrelia burgdoferi DNA in biological samples from patients with sarcoidosis. The target DNA sequence was of chromosomal origin. The amplified DNA sequence was analyzed by agarose gel electrophoresis, PAGE with silver staining, and the identity of amplified DNA was confirmed by restriction enzyme cleavage and DNA-DNA hybridization with a 32P-labelled probe. The assay was sensitive to fewer than two copies of B. burgdorferi genome, even in the presence of a 104-fold excess of human eukaryotic DNA , and was also specific to different B. burgdorferi strains tested. Sera serologically positive to B. burgdcrferi (n=26), bronchoalveolar lavage fluid and supernatant of BALF (n=26) and peripheral blood (n=9) from sarcoidosis patients were tested. The positive rate was low (4/26, 2/26. and 0/9, respectively). It was considered that DNA from B. burgdor ferimay be identified in a minority of patients with sarcoidosis, and it may play a pathogenetic role in such cases. More studies need to be done before advancing the hypothesis of an etiologic role of B. burgdorferi in sarcoidosis.
文摘One oligonueleotide probe,HIV env 101-1,and two oligonucleotide primers,HIV env125-1 and HIV env 125-2,were designed with the aid of a computer and synthesized by a DNAsynthesizer.The env 125 peptide gene was amplified by polymerase chain reaetion(PCR).Beingidentified by electrophoresis and Southern blotting,the PCR product was cloned into plasmidpUC 19.The recombinant pENV 125,identified with X-gal selection and restriction mapping, wassequenced.The results showed that the cloned env 125 peptide gene contained the inserted EcoRⅠ site and ATG at the 5’end,Hind Ⅲ site and TAG at the 3’end.The sequence and readingframe were proved to be correct.
文摘The sensitivity of PCR was determined for detection of HBV DNA in paraffin-embed-ded liver tissue with different methods for sample preparation.Of 8 cases of HBeAg-positiveHBV infection,HBV DNA was detected in 6 by extracting DNA from both frozen and dewaxedsamples,but in none by direct reaction.Of 12 cases subjected to Southern blot hybridization,HBV DNA was detected in 7 by this technique,in 10 by PCR with both methods of DNA extrac-tion and in 3 by direct PCR.The results showed that PCR was sensitive and was comparablewith blot hybridization in detecting intrahepatic HBV DNA.In comparison between differentmethods of sample preparation,the viral detection rate from the dewaxed samples was near thatfrom the frozen ones,while by the direct reaction HBV DNA could be detected only in a fewsamples with high level of infection.
文摘To study the diagnosis of hepatitis C viremia,17 patients(including 14 in acute phase)with post-transfusion non-A,non-B hepatitis(NANBH)were investigated by nested polymerasechain reaction after reverse transcription with 3 sets of primers complemented to different regions ofthe viral sequence.Hepatitis C virus(HCV)RNA was detected in 6(35%)cases with reverse tran-script polymerase chain reaction(RT-PCR)using the primers according to the Japanese clones;in13(76%)using the primers according to the non-coding region which is highly conserved in theviral sequence,and in 16(94%)using both or either one of these two sets of primers.However,no HCV RNA was detected with the primers according to the Chiron prototype.The nucleotide se-quence of HCV is quite variable,and the virus level in blood is very low,therefore,in order to getprecise detection,it is suggested to do nested RT-PCR with several sets of primers complementaryto different conserved regions.
文摘On the basis of the characteristics of Y-chromosome sequence which consists of Y specific repeat DNA family(DYZI) of 800￣5 000 copies,a pair of primers Y3,Y4 is designed to amplify the specific DNA segment of 446 bp by the polymerase chain reaction(PCR),so as to detect the presence of Y chromosome in male fetal cell in maternal peripheral blood for the purpose of prenatal determination of fetal sex.The authors made use of PCR amplification of crude DNA from maternal peripheral blood in early, mid,and late pregnancies to determine the fetal sex.Comparison of the results with those of PCR of their corresponding chorionic villi and amniotic fluid,and with the sex of the aborted fetus or the new born showed coincident rates of 93%,100%,and 87. 5% in early, mid,and late pregnancies respectively.The procedure offers a new alternative way for non-invasive prenatal diagnosis,
文摘in the present study,9 exon-containing DNA segments of dystrophin gene with 9 sets of oligonucleotide primers by two-step multiplex polymerase chain reaction (mPCR) were amplified. Subsequently,gene analysis was performed in 36 cases of Duchenne mascular dystroply (DMD) and 4 cases of Becker muscular dystrophy(BMD). The findings showed that 17 cases of deletion were detected by using the first 5 sets of primers with a relatively high incidence of deletion detection and 2 more cases of deletion were detected by using the remaining 4 sets of primers. The total deletion rate detected by mPCR with 9 cases of primers was 47. 5% of the patients examined,suggesting that about 79. 1% of the patients with gene deletion could be detected. Thus,as a preliminary screening, the two-step mPCR can be used in the gene diagnosis of DMD/BMD. The method is not only simple, convenient and rapid,but also free from radiosotope trouble.
文摘The polymerase chain reaction (PCR) was used for the detection of toxigenic Clostridium difficile (Cd). A primer pair derived from non-repeating sequences of the toxin A gene were used to amplify a 306 bp DNA fragment. Amplified products were visualized by polyacrylamide gel electrophoresis followed by ethidium bromide staining. All 19 strains of toxigenic Cd generated single specific amplified DNA. In contrast, none of the 8 strains of non-toxigenic Cd, 2 strains of C. sordelli and 2 strains of E. coli gave positive results. After the detected DNA of toxigenic Cd was diluted to 0.5ng, the polymerase chain reaction assays were still positive. The results demonstrate that polymerase chain reaction is a simple, rapid, specific and sensitive method for the detection of toxigenic Cd and could be used for the direct detection of Cd in feces samples.
文摘The cDNA fragment of hepatitis C virus (HCV)was obtained by polymerase chain reaction(PCR) using non-coding region primers. Then this fragment was used as a template to produce single strand(SS) HCV cDNA with asymmetric PCR. This sscDNA can be used as template for sequencing directly. The result is in line with that reported by Takamizawa.This method is both simple and convenient,and can be used also in other molecular virological studies.
文摘To assess the presence of HHV-6-Specific DNA in human salivary glands, eighteen specimens of salivary gland tissue were investigated using the polymerase chain reaction. Eight of nine parotid glands, five of seven submandibular glands and one of two sublingual glands were found to have amplification of the HHV-6-specific sequence. The findings suggest that salivary gland tissue is one of the potential sites for HHV-6 persistence following primary infection and that saliva is a vehicle for transmission of the virus.
文摘In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samples of 113 cases of HCC and 69 controls (including 30 cases of liver cirrhosis, 9 cases of metastatic liver cancer and 30 normal subjects). 20/43 (46. 5% ) cases of HCC and 2/30 (6. 7% ) cases of liver cirrhosis are positive and the cases of nletastatic liver cancer and normal controls were negative for human AFP(hAFP) rnRNA. The presence of hAFP mRNA in the peripheral blood seems to be correlated with intrahepatic and distant nletastasls of HCC and portal vein thrombosis. It is concluded that the presence of hAFP mRNA in the peripheral hloocl is an indicator of circulating HCC cells and can be used to diagnose the rnetastasisof HCC through henlatogenous route and RT-PCR amplification of hAFP mRNA is a sensitive and specificprocedure for detecting circulating cells of HCC.
文摘A nested polymerase chain reaction(N-PCR)for the spegific detection of Helicobacterpylori(H.pylori)was developed with two primer pairs(nested primers)derived from ureasegene A of H.pylori.The N-PCR was used to detect 21 different samples of H.pylori including20 clinical isolates and 1 reference strain NCTC 14126,but it was negative for other bacterialspecies,showing the N-PCR assay to be 100% specific.Tenfold serial dilution experiments re-vealed the detection of as little as 0.1 fg of H.pylori DNA by N-PCR.To evaluate the PCR as-say for clinical samples,gastric biopsies were tested with N-PCR,and the results were comparedwith those of culture,urease test and histologic examination(reference standard,RS).In 30biopsy specimens,H.pylori DNA sequences were detected by PCR in all of 20(100%)positivetissue and none of the 10 negative tissues.PCR is a specific and sensitive method that can detectthe presence of H.pylori without the need for culture and would have significant importance di-agnostically and epidemiologically.
文摘目的根据DNA旋转酶B亚基(gyrB)基因设计特异性的引物探针,建立一种能够快速准确鉴定阪崎克罗诺杆菌的实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)方法。方法寻找并在美国国家生物技术信息中心(National Center for Biotechnology Information,NCBI)中下载目标基因序列,使用DNAMAN进行序列比对,Primer Express软件设计引物探针。通过特异性实验、绝对灵敏度、相对灵敏性实验、抗干扰实验对所建立方法进行方法验证。选择本实验室保存的36种40株食品中常见致病菌的标准菌株进行特异性验证。结果多维度特异性验证实验结果显示该方法能够特异性地检测出阪崎克罗诺杆菌,对亲缘关系较近的其他克罗诺杆菌及食品中较为常见的致病菌均无非特异性扩增。DNA检测灵敏度可以达到0.0100 ng/μL,相对灵敏度可以达到10^(3) CFU/mL。抗干扰实验结果显示,将干扰菌和干扰菌DNA分别与阪崎克罗诺杆菌和阪崎克罗诺杆菌DNA进行混合检测,对检测结果无显著影响,说明该方法抗干扰能力良好。结论本研究所设计的引物探针在实时荧光PCR方法下对食品样品中阪崎克罗诺杆菌的检测具有特异、快速、敏感和抗干扰的特点,可为以后食品中阪崎克罗诺杆菌的检测提供技术支撑。
