AIM:To develop a real-time polymerase chain reaction(PCR) method to detect and quantify Campylobacter jejuni(C.jejuni) from stool specimens.METHODS:Primers and a probe for real-time PCR were designed based on the spec...AIM:To develop a real-time polymerase chain reaction(PCR) method to detect and quantify Campylobacter jejuni(C.jejuni) from stool specimens.METHODS:Primers and a probe for real-time PCR were designed based on the specific DNA sequence of the hipO gene in C.jejuni.The specificity of the primers and probe were tested against a set of Campylobacter spp.and other enteric pathogens.The optimal PCR conditions were determined by testing a series of conditions with standard a C.jejuni template.The detection limits were obtained using purified DNA from bacterial culture and extracted DNA from the stool specimen.Two hundred and forty-two specimens were analyzed for the presence of C.jejuni by direct bacterial culture and real-time PCR.RESULTS:The optimal PCR system was determined using reference DNA templates,1 × uracil-DNA glycosylase,3.5 mmol/L MgCl 2,1.25 U platinum Taq polymerase,0.4 mmol/L PCR nucleotide mix,0.48 μmol/L of each primer,0.2 μmol/L of probe and 2 μL of DNA template in a final volume of 25 μL.The PCR reaction was carried as follows:95 ℃ for 4 min,followed by 45 cycles of 10 s at 95 ℃ and 30 s at 59 ℃.The detection limit was 4.3 CFU/mL using purified DNA from bacterial culture and 10 3 CFU/g using DNA from stool specimens.Twenty(8.3%,20/242) C.jejuni strains were isolated from bacterial culture,while 41(16.9%,41/242) samples were found to be positive by realtime PCR.DNA sequencing of the PCR product indicated the presence of C.jejuni in the specimen.One mixed infection of C.jejuni and Salmonella was detected in one specimen and the PCR test for this specimen was positive.CONCLUSION:The sensitivity of detection of C.jejuni from stool specimens was much higher using this PCR assay than using the direct culture method.展开更多
Objective:To compare the sensitivity and specificity of direct fecal smear microscopy,culture,and polymerase chain reaction in the detection of Blastocystis sp.in human stool.Methods:Human stool samples were collected...Objective:To compare the sensitivity and specificity of direct fecal smear microscopy,culture,and polymerase chain reaction in the detection of Blastocystis sp.in human stool.Methods:Human stool samples were collected from a community in San Isidro,Rodriguez,Rizal,Philippines.These samples were subjected to direct fecal smear microscopy,culture and polymerase chain reaction to detect the presence of Blastocystis sp.Results:Of the 110 stool samples collected,28(25%)were detected positive for the presence of Blastocystis sp.by two or more tests.Culture method detected the highest number of Blastocystis-positive stool samples(n=36),followed by PCR of DNA extracted from culture(n=26),PCR of DNA extracted from stool(n=10),and direct fecal smear(n=9).Compared to culture,the sensitivity of the other detection methods were 66.7%for PCR from culture and 19.4%for both PCR from stool and direct fecal smear.Specificity of the methods was high,with PCR from culture and direct fecal smear having97.3%,while PCR from stool at 95.9%.Conclusions:In this study,in vitro culture is the best method for detecting Blastocystis sp.in human stool samples.展开更多
White piedra is a superficial, chronic, asymptomatic mycoses caused by yeast fungi of the genus Trichosporon. It affects the hair, especially of the head, less frequently of the pubis, perineum, armpit, beard, mustach...White piedra is a superficial, chronic, asymptomatic mycoses caused by yeast fungi of the genus Trichosporon. It affects the hair, especially of the head, less frequently of the pubis, perineum, armpit, beard, mustache, eyebrows and eyelashes, and is characterized by the formation of soft nodules or fungal clusters. It affects all age groups and both sexes, predominantly women. Diagnosis is made by direct examination of the affected hair and culture on Sabouraud dextrose agar. The identification of species occurs through more specific identification procedures, such as mass spectrometry (MALDI-TOF) and PCR). The objective of this work is to report two cases of familial white piedra caused by T. inkin identified by PCR.展开更多
Tomato seedlings damping-off is a limiting factor in commercial greenhouse production. To determine the causal agents of disease, sampling and fungal isolation were performed during 2012. Samples were collected from i...Tomato seedlings damping-off is a limiting factor in commercial greenhouse production. To determine the causal agents of disease, sampling and fungal isolation were performed during 2012. Samples were collected from infected seedlings growing in greenhouses in the Syrian coastal region. Isolation of fungi was done in the laboratories of the Agronomical Reaserch Center, in Lattakia and the molecular analyses were done in the Biotechnology Center at Tishreen University, Lattakia, Syria, during the years 2012, 2013. Eight isolates ofPythium sp. obtained were purified using hyphal tip method （named P1, P2, P3, P4, P5, P6, P7 and P8）. Isolates were morphologically identified by optical microscope, then molecularly Characterized using genus specific ITS primers. The results of morphological characterization of pathogenic species suggested the detection of Pythium aphanidermatum, P. ultimum. The analysis of DNAs from the different isolates with ITS primers, recognizing the inter transcript spacer of nuclear ribosomal DNA proved that the eight, isolates were belonging to the species P. ultimum. The complete sequences of ribosomal DNA internal transcribed spacers regions of selected isolates were determined and submitted to GenBank. The GenBank-BLAST homology search revealed P. ultimum as the most similar sequence （〉 96% identity） with GenBank entry AB355596.展开更多
作为影响全球变化的主要因子之一,氮沉降对生态系统生物地球化学循环有重要的影响。细菌作为土壤生态系统物质循环的关键参与者,是土壤生态系统变化的敏感指标,在氮沉降对生态系统影响过程中发挥不可忽视的作用。模拟2种施氮方式(SAN:...作为影响全球变化的主要因子之一,氮沉降对生态系统生物地球化学循环有重要的影响。细菌作为土壤生态系统物质循环的关键参与者,是土壤生态系统变化的敏感指标,在氮沉降对生态系统影响过程中发挥不可忽视的作用。模拟2种施氮方式(SAN:土表施氮,LAN:叶面施氮)和3种施氮量(5.6、15.6、20.6 g N m^(-2)a^(-1)),运用PCR-DGGE技术,分析不同施氮方式和施氮量对两种盆栽植物马尾松(Pinus massoniana Lamb.)和木荷(Schima superba Gardn.et Champ.)幼苗土壤细菌多样性和群落组成的影响。结果表明:不同氮添加方式下,土壤细菌多样性和群落组成对氮添加的响应不同,并受到季节的影响。在雨季,LAN处理木荷土壤细菌多样性高于SAN处理;而旱季,LAN处理两种幼苗根系土壤细菌多样性均高于SAN处理。LAN处理条件下,马尾松土壤AcidobacteriaGp1相对丰度在旱季和雨季均显著高于SAN处理(P<0.05)。在雨季,马尾松LAN处理土壤Alphaproteobacteria相对丰度显著低于SAN处理(P<0.05),旱季没有显著差异(P>0.05)。氮添加能够提高木荷土壤细菌多样性,提高马尾松土壤变形菌门的相对丰度。旱季施氮还能够提高马尾松土壤Actinobacteria相对丰度,降低木荷土壤酸杆菌门的相对丰度。土壤细菌群落变化与土壤pH显著相关(P<0.05),木荷土壤细菌群落还受到土壤NH+4-N含量的显著影响(P<0.05)。展开更多
基金Supported by The General Program of National Natural Science Foundation of China,No.81271789the Major State Basic Research Development Program,No.2013CB127204
文摘AIM:To develop a real-time polymerase chain reaction(PCR) method to detect and quantify Campylobacter jejuni(C.jejuni) from stool specimens.METHODS:Primers and a probe for real-time PCR were designed based on the specific DNA sequence of the hipO gene in C.jejuni.The specificity of the primers and probe were tested against a set of Campylobacter spp.and other enteric pathogens.The optimal PCR conditions were determined by testing a series of conditions with standard a C.jejuni template.The detection limits were obtained using purified DNA from bacterial culture and extracted DNA from the stool specimen.Two hundred and forty-two specimens were analyzed for the presence of C.jejuni by direct bacterial culture and real-time PCR.RESULTS:The optimal PCR system was determined using reference DNA templates,1 × uracil-DNA glycosylase,3.5 mmol/L MgCl 2,1.25 U platinum Taq polymerase,0.4 mmol/L PCR nucleotide mix,0.48 μmol/L of each primer,0.2 μmol/L of probe and 2 μL of DNA template in a final volume of 25 μL.The PCR reaction was carried as follows:95 ℃ for 4 min,followed by 45 cycles of 10 s at 95 ℃ and 30 s at 59 ℃.The detection limit was 4.3 CFU/mL using purified DNA from bacterial culture and 10 3 CFU/g using DNA from stool specimens.Twenty(8.3%,20/242) C.jejuni strains were isolated from bacterial culture,while 41(16.9%,41/242) samples were found to be positive by realtime PCR.DNA sequencing of the PCR product indicated the presence of C.jejuni in the specimen.One mixed infection of C.jejuni and Salmonella was detected in one specimen and the PCR test for this specimen was positive.CONCLUSION:The sensitivity of detection of C.jejuni from stool specimens was much higher using this PCR assay than using the direct culture method.
基金supported by a research grant from the Office of the Vice-Chancellor for Research and Development,University of the Philippines-Diliman(Grant No.101007 PNSE)to W.L.R.and H.J.S
文摘Objective:To compare the sensitivity and specificity of direct fecal smear microscopy,culture,and polymerase chain reaction in the detection of Blastocystis sp.in human stool.Methods:Human stool samples were collected from a community in San Isidro,Rodriguez,Rizal,Philippines.These samples were subjected to direct fecal smear microscopy,culture and polymerase chain reaction to detect the presence of Blastocystis sp.Results:Of the 110 stool samples collected,28(25%)were detected positive for the presence of Blastocystis sp.by two or more tests.Culture method detected the highest number of Blastocystis-positive stool samples(n=36),followed by PCR of DNA extracted from culture(n=26),PCR of DNA extracted from stool(n=10),and direct fecal smear(n=9).Compared to culture,the sensitivity of the other detection methods were 66.7%for PCR from culture and 19.4%for both PCR from stool and direct fecal smear.Specificity of the methods was high,with PCR from culture and direct fecal smear having97.3%,while PCR from stool at 95.9%.Conclusions:In this study,in vitro culture is the best method for detecting Blastocystis sp.in human stool samples.
文摘White piedra is a superficial, chronic, asymptomatic mycoses caused by yeast fungi of the genus Trichosporon. It affects the hair, especially of the head, less frequently of the pubis, perineum, armpit, beard, mustache, eyebrows and eyelashes, and is characterized by the formation of soft nodules or fungal clusters. It affects all age groups and both sexes, predominantly women. Diagnosis is made by direct examination of the affected hair and culture on Sabouraud dextrose agar. The identification of species occurs through more specific identification procedures, such as mass spectrometry (MALDI-TOF) and PCR). The objective of this work is to report two cases of familial white piedra caused by T. inkin identified by PCR.
文摘Tomato seedlings damping-off is a limiting factor in commercial greenhouse production. To determine the causal agents of disease, sampling and fungal isolation were performed during 2012. Samples were collected from infected seedlings growing in greenhouses in the Syrian coastal region. Isolation of fungi was done in the laboratories of the Agronomical Reaserch Center, in Lattakia and the molecular analyses were done in the Biotechnology Center at Tishreen University, Lattakia, Syria, during the years 2012, 2013. Eight isolates ofPythium sp. obtained were purified using hyphal tip method （named P1, P2, P3, P4, P5, P6, P7 and P8）. Isolates were morphologically identified by optical microscope, then molecularly Characterized using genus specific ITS primers. The results of morphological characterization of pathogenic species suggested the detection of Pythium aphanidermatum, P. ultimum. The analysis of DNAs from the different isolates with ITS primers, recognizing the inter transcript spacer of nuclear ribosomal DNA proved that the eight, isolates were belonging to the species P. ultimum. The complete sequences of ribosomal DNA internal transcribed spacers regions of selected isolates were determined and submitted to GenBank. The GenBank-BLAST homology search revealed P. ultimum as the most similar sequence （〉 96% identity） with GenBank entry AB355596.
文摘作为影响全球变化的主要因子之一,氮沉降对生态系统生物地球化学循环有重要的影响。细菌作为土壤生态系统物质循环的关键参与者,是土壤生态系统变化的敏感指标,在氮沉降对生态系统影响过程中发挥不可忽视的作用。模拟2种施氮方式(SAN:土表施氮,LAN:叶面施氮)和3种施氮量(5.6、15.6、20.6 g N m^(-2)a^(-1)),运用PCR-DGGE技术,分析不同施氮方式和施氮量对两种盆栽植物马尾松(Pinus massoniana Lamb.)和木荷(Schima superba Gardn.et Champ.)幼苗土壤细菌多样性和群落组成的影响。结果表明:不同氮添加方式下,土壤细菌多样性和群落组成对氮添加的响应不同,并受到季节的影响。在雨季,LAN处理木荷土壤细菌多样性高于SAN处理;而旱季,LAN处理两种幼苗根系土壤细菌多样性均高于SAN处理。LAN处理条件下,马尾松土壤AcidobacteriaGp1相对丰度在旱季和雨季均显著高于SAN处理(P<0.05)。在雨季,马尾松LAN处理土壤Alphaproteobacteria相对丰度显著低于SAN处理(P<0.05),旱季没有显著差异(P>0.05)。氮添加能够提高木荷土壤细菌多样性,提高马尾松土壤变形菌门的相对丰度。旱季施氮还能够提高马尾松土壤Actinobacteria相对丰度,降低木荷土壤酸杆菌门的相对丰度。土壤细菌群落变化与土壤pH显著相关(P<0.05),木荷土壤细菌群落还受到土壤NH+4-N含量的显著影响(P<0.05)。
