Objective:To identify members of genera of rickettsia and O.tsutsugammhi simultaneously.Methods:Rapid and duplex and nested PCR methods have been established by designing primers based on the conserved regions of heat...Objective:To identify members of genera of rickettsia and O.tsutsugammhi simultaneously.Methods:Rapid and duplex and nested PCR methods have been established by designing primers based on the conserved regions of heat shock protein GroEL gene.345 mouse viscera samples including liver,spleen and kidney,96 Xenopsylla cheopis and 32 chiggers collected from Hongta areas of Yuxi city,Yunnan province were tested by the new PCR methods.Results:The result of the study showed that the new PCR methods could identify most members of genera -Rickettsia and Orientia simultaneously with 100%specificity and its sensitivity could test one copy per microliter.The results of detection prevalence of rickettsioses in mouse,flea and mites DNA samples showed that the total rickettsia infection rate in mouse was 34.78%(120/345).The total infection rates in R.typhi,O.t Karp and R.sibirica of mouse samples were 28.12%(97/345),19.71%(68/345) and O. 29%(1/345) respectively.Co-infection rates in R.typhi and 0.t Karp of mouse samples were 13.33%(46/ 345).O.t Karp type has been the main epidemic strain in these areas.Conclusion:We concluded that this PCR method could be used to detect multi-genera rickettsia simultaneously.Molecular evidences provided in this and previous studies strongly support that Hongta areas of Yuxi city are a natural focus for typhus and scrub typhus with the common occurrence of their confection.展开更多
Objective:To design a duplex PCR for rapid and simultaneous detection of Brucella species, in human blood samples.Methods:Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis.Fo...Objective:To design a duplex PCR for rapid and simultaneous detection of Brucella species, in human blood samples.Methods:Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis.Following DNA extraction,PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals.Results:Of the 52 peripheral bloods samples tested, 25 sample(48%) showed positive reactions in PCR.Twelve samples were positive for Brucella abortus(B.abortus)(23%,13 for Brucella melUensis(B.melUensis)(25%) and 0 for Brucella ovis (6.ovis)(Ow.Conclusions:This work de=monstrates dial in case where specific primers were utilized,duplex PCR has proved to be a simple,fast,and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples.展开更多
Myotonic dystrophy type 1 (DM1),or Steiner’s disease,is an autosomal dominant disorder caused by the expansion of unstable trinucleotide repeats (CTG) in the 3’ untranslated region of the myotonic dystrophy protein ...Myotonic dystrophy type 1 (DM1),or Steiner’s disease,is an autosomal dominant disorder caused by the expansion of unstable trinucleotide repeats (CTG) in the 3’ untranslated region of the myotonic dystrophy protein kinase gene (DMPK) (Brook et al.,1992;Mahadevan et al.,1992).The number of CTG repeats observed in normal individuals is in a range of 5-34,while the individuals with 35-49 CTG repeats are usually asymptomatic but at risk of展开更多
Recently, the incidence of<span> </span><i><span>Candida</span></i><span> infections has substantially increased. Conventional identification methods for </span><...Recently, the incidence of<span> </span><i><span>Candida</span></i><span> infections has substantially increased. Conventional identification methods for </span><i><span>Candida</span></i><span> species are technically difficult to conduct and cannot accurately distinguish each species. The purpose of the present study was to design primers to identify and detect simultaneously</span><span> </span><span>eight medically important </span><i><span>Candida</span></i><span> species using one-step multiplex PCR. PCR primers were designed based on partial sequences of intergenic spacer (IGS) and internal transcribed spacer (ITS) genes of eight medically important </span><i><span>Candida</span></i><span> species. These primers were able to distinguish each </span><i><span>Candida</span></i><span> species and did not display cross-reactivity with representative </span><i><span>Candida </span></i><span>species other than the eight</span><i><span> Candida</span></i><span> species. Moreover, our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.</span>展开更多
H9 s ubtype avian influenza virus（AIV） and infectious bronchitis virus（IBV） are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg producti...H9 s ubtype avian influenza virus（AIV） and infectious bronchitis virus（IBV） are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction（RT-PCR） assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR（d RT-PCR） was established. Two primer sets target the hemagglutinin（HA） gene of H9 AIV and the nucleocapsid（N） gene of IBV, respectively. Spec ific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the d RT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×10^1, 1.5×10^1 and 1.5×10^1 50% egg infective doses（EID_（50）） m L^–1, respectively. The concordance rates between the d RT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the d RT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and survei llance of H9 AIVs and IBVs.展开更多
Objective: To develop a duplex real-time PCR assay for pharmaceutical rapid microbial detection of Staphylococcus aureus and Pseudomonas aeruginosa. Methods: The specific primers and probes were designed to amplify th...Objective: To develop a duplex real-time PCR assay for pharmaceutical rapid microbial detection of Staphylococcus aureus and Pseudomonas aeruginosa. Methods: The specific primers and probes were designed to amplify the femB gene of S. aureus and the DNA gyrase subunit B gene of P. aeruginosa. The sensitivity of the system was detected by a multiple proportional dilution method. In order to examine the specificity of the system, other twenty-one bacteria strains were assayed simultaneously. Results: A highly sensitive and specific duplex real-time PCR assay for the detection of S. aureus and P. aeruginosa was established. The sensitivity was 50 copies/μL. The specificity was 100%. The whole detection procedure can be finished within 2.5 h. Conclusion: The duplex real-time PCR method is efficient in detecting with good sensitivity and specificity. There is a good prospect of this method applying in disease prevention and pharmaceutical industry due to the simultaneous detection of two pathogens.展开更多
[ Objective] To improve the accuracy and efficiency of the detection which used in Perkinsus sp and Marteilia refringens, and then short- en the detective cycle. [ Method] According to the gene sequence of Perkinsus s...[ Objective] To improve the accuracy and efficiency of the detection which used in Perkinsus sp and Marteilia refringens, and then short- en the detective cycle. [ Method] According to the gene sequence of Perkinsus sp and Marteilia refringens from gene bank, design two pairs of spe- cific primers and two TaqMan probes with different fluorophores labeled. Optimizing the reactive conditions and reagent concentration in order that establishing the duplex real-time PCR method for detecting Perkinsus sp and Marteilia refringens simultaneously. [ Result ] The sensitivity of the du- plex real-time PCR method which about Pertdnsus sp and Marteilia refringens is 40 template copies. After combine the templates of Perkinsus sp and Marteilia refringens with different concentrations, this method still could be detect this two protozoan efficiently and synchronously. [ Condudon] The es- tablished duplex real-time PCR method for detecting Perkinsus sp. and Marteilia refringens possesses lots of advantages, such as specific, sensitive, rapid, quantitative and reproducible, can be used for clinical detection of infection which was caused by Perkinsus sp. and Marteilia refringens.展开更多
The paper aimed to establish a duplex PCR method for simultaneous detection of Pseudomonas savastanoi pv. Phaseolicola （Psp） and Curtobaeterium /accumfadens pv. Flaccumfaciens （Cff）. Based on the argK gene of Psp ...The paper aimed to establish a duplex PCR method for simultaneous detection of Pseudomonas savastanoi pv. Phaseolicola （Psp） and Curtobaeterium /accumfadens pv. Flaccumfaciens （Cff）. Based on the argK gene of Psp in GenBank, the primers PSPF1/PSPR2 were designed. The duplex PCR assay was dereloped using the combined primers PSPF1/PSPR2 and CflF1/CffR2, which were specific primers for Cff. The reaction conditions were optimized and specificity md sensitivity of the duplex PCR were tested. The expected DNA fragment was specifically amplified from the genomic DNA of Psp and Cff. Specificity was conirmed in the artificially inoculated soybean samples imparted. Thus, the duplex PCR developed in this study could be used for the simultaneous detection of Psp md Cff from imported soybean.展开更多
[Objective]The paper was to establish a one-step reverse transcriptase droplet digital PCR(RT-ddPCR)assay for bovine viral diarrhea virus(BVDV).[Method]Based on one-step real-time quantitative PCR(RT-qPCR)assay,BVDV-s...[Objective]The paper was to establish a one-step reverse transcriptase droplet digital PCR(RT-ddPCR)assay for bovine viral diarrhea virus(BVDV).[Method]Based on one-step real-time quantitative PCR(RT-qPCR)assay,BVDV-specific primers and probes were designed in this study.The reverse transcriptase,annealing temperature,primer and probe concentrations and reaction conditions of RT-ddPCR assay were optimized.Meantime,the specificity,sensitivity and repeatability of RT-ddPCR assay were evaluated.[Result]The optimal reverse transcription system for the established RT-ddPCR assay was as follows:commercial one-step reverse transcriptase droplet digital PCR kit with matching reagents,a final primer concentration of 900 nmol/L,a final probe concentration of 250 nmol/L and an optimal annealing temperature of 57℃.The results were negative when the method was used to detect other common epidemic viruses;the minimum detection limit was 3.2 copies/μL with good repeatability,and the coefficient of variation was less than 5%.RT-ddPCR and RT-qPCR assays were used to test 24 bovine swab samples and the test results showed that the established RT-ddPCR assay was superior to RT-qPCR assay.[Conclusion]The RT-ddPCR assay established in this study has strong specificity,high sensitivity and good repeatability,and is suitable for nucleic acid detection of clinical samples.This study provided a technical support for early detection and quantitative diagnosis of BVDV infection.展开更多
基金supported by National Natural Science Foundation of China(No. 30771854)China-U.S.Collaborative Program on Emerging and Re-emerging Infectious Diseases(No. 1U2GGH000018-01)
文摘Objective:To identify members of genera of rickettsia and O.tsutsugammhi simultaneously.Methods:Rapid and duplex and nested PCR methods have been established by designing primers based on the conserved regions of heat shock protein GroEL gene.345 mouse viscera samples including liver,spleen and kidney,96 Xenopsylla cheopis and 32 chiggers collected from Hongta areas of Yuxi city,Yunnan province were tested by the new PCR methods.Results:The result of the study showed that the new PCR methods could identify most members of genera -Rickettsia and Orientia simultaneously with 100%specificity and its sensitivity could test one copy per microliter.The results of detection prevalence of rickettsioses in mouse,flea and mites DNA samples showed that the total rickettsia infection rate in mouse was 34.78%(120/345).The total infection rates in R.typhi,O.t Karp and R.sibirica of mouse samples were 28.12%(97/345),19.71%(68/345) and O. 29%(1/345) respectively.Co-infection rates in R.typhi and 0.t Karp of mouse samples were 13.33%(46/ 345).O.t Karp type has been the main epidemic strain in these areas.Conclusion:We concluded that this PCR method could be used to detect multi-genera rickettsia simultaneously.Molecular evidences provided in this and previous studies strongly support that Hongta areas of Yuxi city are a natural focus for typhus and scrub typhus with the common occurrence of their confection.
基金supported by Molecular Biology Research Center, Baqiyatallah University of Medical Sciences,with grant number BMSU/MBRC-90-001
文摘Objective:To design a duplex PCR for rapid and simultaneous detection of Brucella species, in human blood samples.Methods:Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis.Following DNA extraction,PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals.Results:Of the 52 peripheral bloods samples tested, 25 sample(48%) showed positive reactions in PCR.Twelve samples were positive for Brucella abortus(B.abortus)(23%,13 for Brucella melUensis(B.melUensis)(25%) and 0 for Brucella ovis (6.ovis)(Ow.Conclusions:This work de=monstrates dial in case where specific primers were utilized,duplex PCR has proved to be a simple,fast,and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples.
基金supported by the National Basic Research Program of China(2010CB529601 and 2013CB945404)to B.L.Wuthe Fudan Young Teacher Funding to Y.An,the higher Education Research Project of Gansu Province(2015B-094)to X.LanShanghai Children’s Hospital Funding(2016YMS001)to X.Lan
文摘Myotonic dystrophy type 1 (DM1),or Steiner’s disease,is an autosomal dominant disorder caused by the expansion of unstable trinucleotide repeats (CTG) in the 3’ untranslated region of the myotonic dystrophy protein kinase gene (DMPK) (Brook et al.,1992;Mahadevan et al.,1992).The number of CTG repeats observed in normal individuals is in a range of 5-34,while the individuals with 35-49 CTG repeats are usually asymptomatic but at risk of
文摘Recently, the incidence of<span> </span><i><span>Candida</span></i><span> infections has substantially increased. Conventional identification methods for </span><i><span>Candida</span></i><span> species are technically difficult to conduct and cannot accurately distinguish each species. The purpose of the present study was to design primers to identify and detect simultaneously</span><span> </span><span>eight medically important </span><i><span>Candida</span></i><span> species using one-step multiplex PCR. PCR primers were designed based on partial sequences of intergenic spacer (IGS) and internal transcribed spacer (ITS) genes of eight medically important </span><i><span>Candida</span></i><span> species. These primers were able to distinguish each </span><i><span>Candida</span></i><span> species and did not display cross-reactivity with representative </span><i><span>Candida </span></i><span>species other than the eight</span><i><span> Candida</span></i><span> species. Moreover, our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.</span>
基金supported by the National High-Tech R&D Program of China(2012AA101303)
文摘H9 s ubtype avian influenza virus（AIV） and infectious bronchitis virus（IBV） are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction（RT-PCR） assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR（d RT-PCR） was established. Two primer sets target the hemagglutinin（HA） gene of H9 AIV and the nucleocapsid（N） gene of IBV, respectively. Spec ific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the d RT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×10^1, 1.5×10^1 and 1.5×10^1 50% egg infective doses（EID_（50）） m L^–1, respectively. The concordance rates between the d RT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the d RT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and survei llance of H9 AIVs and IBVs.
文摘Objective: To develop a duplex real-time PCR assay for pharmaceutical rapid microbial detection of Staphylococcus aureus and Pseudomonas aeruginosa. Methods: The specific primers and probes were designed to amplify the femB gene of S. aureus and the DNA gyrase subunit B gene of P. aeruginosa. The sensitivity of the system was detected by a multiple proportional dilution method. In order to examine the specificity of the system, other twenty-one bacteria strains were assayed simultaneously. Results: A highly sensitive and specific duplex real-time PCR assay for the detection of S. aureus and P. aeruginosa was established. The sensitivity was 50 copies/μL. The specificity was 100%. The whole detection procedure can be finished within 2.5 h. Conclusion: The duplex real-time PCR method is efficient in detecting with good sensitivity and specificity. There is a good prospect of this method applying in disease prevention and pharmaceutical industry due to the simultaneous detection of two pathogens.
文摘[ Objective] To improve the accuracy and efficiency of the detection which used in Perkinsus sp and Marteilia refringens, and then short- en the detective cycle. [ Method] According to the gene sequence of Perkinsus sp and Marteilia refringens from gene bank, design two pairs of spe- cific primers and two TaqMan probes with different fluorophores labeled. Optimizing the reactive conditions and reagent concentration in order that establishing the duplex real-time PCR method for detecting Perkinsus sp and Marteilia refringens simultaneously. [ Result ] The sensitivity of the du- plex real-time PCR method which about Pertdnsus sp and Marteilia refringens is 40 template copies. After combine the templates of Perkinsus sp and Marteilia refringens with different concentrations, this method still could be detect this two protozoan efficiently and synchronously. [ Condudon] The es- tablished duplex real-time PCR method for detecting Perkinsus sp. and Marteilia refringens possesses lots of advantages, such as specific, sensitive, rapid, quantitative and reproducible, can be used for clinical detection of infection which was caused by Perkinsus sp. and Marteilia refringens.
基金Supported by Science and Technology Project of AQSIQ(2013IK277)National Rice Industry System Development Program
文摘The paper aimed to establish a duplex PCR method for simultaneous detection of Pseudomonas savastanoi pv. Phaseolicola （Psp） and Curtobaeterium /accumfadens pv. Flaccumfaciens （Cff）. Based on the argK gene of Psp in GenBank, the primers PSPF1/PSPR2 were designed. The duplex PCR assay was dereloped using the combined primers PSPF1/PSPR2 and CflF1/CffR2, which were specific primers for Cff. The reaction conditions were optimized and specificity md sensitivity of the duplex PCR were tested. The expected DNA fragment was specifically amplified from the genomic DNA of Psp and Cff. Specificity was conirmed in the artificially inoculated soybean samples imparted. Thus, the duplex PCR developed in this study could be used for the simultaneous detection of Psp md Cff from imported soybean.
基金Supported by Science and Technology Development Plan Fund Project of Jilin Province(20210202101NC)YDZJ202203C G Z H 050.
文摘[Objective]The paper was to establish a one-step reverse transcriptase droplet digital PCR(RT-ddPCR)assay for bovine viral diarrhea virus(BVDV).[Method]Based on one-step real-time quantitative PCR(RT-qPCR)assay,BVDV-specific primers and probes were designed in this study.The reverse transcriptase,annealing temperature,primer and probe concentrations and reaction conditions of RT-ddPCR assay were optimized.Meantime,the specificity,sensitivity and repeatability of RT-ddPCR assay were evaluated.[Result]The optimal reverse transcription system for the established RT-ddPCR assay was as follows:commercial one-step reverse transcriptase droplet digital PCR kit with matching reagents,a final primer concentration of 900 nmol/L,a final probe concentration of 250 nmol/L and an optimal annealing temperature of 57℃.The results were negative when the method was used to detect other common epidemic viruses;the minimum detection limit was 3.2 copies/μL with good repeatability,and the coefficient of variation was less than 5%.RT-ddPCR and RT-qPCR assays were used to test 24 bovine swab samples and the test results showed that the established RT-ddPCR assay was superior to RT-qPCR assay.[Conclusion]The RT-ddPCR assay established in this study has strong specificity,high sensitivity and good repeatability,and is suitable for nucleic acid detection of clinical samples.This study provided a technical support for early detection and quantitative diagnosis of BVDV infection.
