Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfl...Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology,and screened multiple AMR genes in broiler fecal samples.Methods A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection.A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.Results The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction.The sensitivity rate,specificity rate,positive predictive value,negative predictive value and correct indices were 99.30%,98.08%,95.31%,99.79%,and 0.9755,respectively.Utilizing this assay,we demonstrate that AMR genes are widely spread,with positive detection rates ranging from 0 to 97.07%in 273 broiler fecal samples.bla CTX-M,bla TEM,mcr-1,fex A,cfr,optr A,and int I1 showed over 80%prevalence.The dissemination of AMR genes was distinct between the two farms.Conclusions We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples.The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.展开更多
With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 ...With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 and spread quickly across the country.It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.Atypical porcine pestivirus(APPV) was first detected in Guangdong Province,China,in 2016,which mainly harms piglets and has a local epidemic situation in southern China.These three diseases have similar clinical symptoms in pig herds,which cause considerable losses to the pig industry.They are difficult to be distinguished only by clinical diagnosis.Therefore,developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.In this study,three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV(5’ UTR),African swine fever virus(ASFV)(B646L),and APPV(5’ UTR),followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.The results showed that the method did not cross-react with other swine pathogens(porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),foot-and-mouth disease virus(FMDV),pseudorabies virus(PRV),porcine parvovirus(PPV),and bovine viral diarrhea virus BVDV).The sensitivity results showed that CSFV,ASFV,and APPV could be detected as low as 1 copy μL–1;the repeatability results showed that the intra-assay and interassay coefficient of variation of ASFV,CSFV,and APPV was less than 1%.Twenty-two virus samples were detected by the multiplex real-time PCR,compared with national standard diagnostic and patented method assay for CSF(GB/T 27540–2011),ASF(GB/T 18648–2020),and APPV(CN108611442A),respectively.The sensitivity of this triple real-time PCR for CSFV,ASFV,and APPV was almost the same,and the compliance results were the same(100%).A total of 451 clinical samples were detected,and the results showed that the positive rates of CSFV,ASFV,and APPV were 0.22% (1/451),1.3%(6/451),and 0%(0/451),respectively.This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV,ASFV,and APPV.展开更多
Objective To describe correlation between multiple genetic tumor markers,carcinoembryonic antigen (CEA),cytokeratin 20 (CK20),and Survivin,and clinicopathological features of colorectal cancer (CRC) and to assess prog...Objective To describe correlation between multiple genetic tumor markers,carcinoembryonic antigen (CEA),cytokeratin 20 (CK20),and Survivin,and clinicopathological features of colorectal cancer (CRC) and to assess prognostic diagnosis value in cancer recurrence and metastasis.Methods A total of 92 patients with CRC,68 patients with precancerous lesions,and 29 control volunteers were collected for the detection of CEA,CK20,and Survivin expressions by using quantitative Real-Time PCR technology.Associations among these measurements and clinicopathological features of CRC,and cancer recurrence and metastasis rates in 4-year follow-up were analyzed.Results No mRNA expressions of CEA,CK20,or Survivin were detected in the control group.Expressions of CEA,CK20,and Survivin were 41.3%,47.8%,and 72.8% in CRC patients,respectively.The expressions of genetic tumor markers were related to the clinical stage and lymph node metastasis.In patients with Survivin high expression,4-year survival rate was significantly lower than that in Survivin low expression.The multiple tumor markers assay for CRC patients showed higher specificity and positive detection rate than single marker assay.Patients with CEA,CK20,and Survivin simultaneous expressions had significantly higher 4-year recurrence rate and death rate than those with only one or two markers expression.ConclusionMultiple tumor markers assay including CEA,CK20,and Survivin in peripheral blood by quantitative Real-Time PCR can be an ideal method for the surveillance of the recurrence and prognosis for CRC patients.展开更多
In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM ric...In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM rice. And the identification of GM rice is of great significance. Therefore, in the present study, the po- tential problems in the identification of GM rice with PCR were analyzed both at a technical level and from a theoretical perspective. In addition, PCR detection on the transgenic elements： promoter, terminator, internal reference gene and target gene was discussed, respectively. The possible solutions were proposed based on the principles of plant virology and genetic engineering.展开更多
Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resi...Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resistance necessitates updating diagnostic techniques to ensure higher sensitivity and specificity, especially with advancements in science and medicine. This study aimed to evaluate the prevalence of UTIs and antibiotic resistance profiles through urine culture, as well as to identify Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp. in urine samples using a molecular approach with multiplex real-time PCR. From May 3 to July 25, 2023, at the Pietro Annigoni Biomolecular Research Center (CERBA) and Saint Camille Hospital of Ouagadougou (HOSCO), 209 urine samples collected from patients with suspected UTIs were analyzed using both urine culture and multiplex real-time PCR. Among the 209 patients, 52.15% were male and 47.85% female, with an average age of 46.87 ± 21.33 years. Urine cultures revealed an overall UTI prevalence of 23.44%, with a prevalence of 8.13% in men versus 15.31% in women (P = 0.023). The bacterial prevalence rates were as follows: Escherichia coli (12.92%), Klebsiella spp. (7.18%), Enterobacter cloacae (1.44%), Staphylococcus aureus (0.96%), and other bacteria. Klebsiella spp. demonstrated 100% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, while Escherichia coli showed 96.2% and 65.4% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, respectively. PCR analysis of the target bacteria revealed mono-infection prevalence rates of Klebsiella pneumoniae (10.39%), Klebsiella oxytoca (7.79%), and Acinetobacter spp. (7.79%), along with a co-infection prevalence rate of Klebsiella pneumoniae/Acinetobacter spp. (1.30%). This study demonstrated that PCR, with its high sensitivity and specificity, could effectively distinguish Klebsiella pneumoniae from Klebsiella oxytoca and detect Acinetobacter spp. in less than 24 hours—something urine culture alone could not achieve. The relative ease of automating urine PCR testing, combined with its diagnostic accuracy and rapid turnaround time, makes it a valuable addition to modern medical practice for the laboratory diagnosis of UTIs.展开更多
应用SYBR Green I染料能选择性结合双链DNA的特点,可检测到沙门氏菌fimI基因特异性靶序列扩增所产生的荧光信号,通过熔解曲线可知其熔点值约为85.6℃,而对其他非沙门氏菌则检测不到荧光信号。建立了一种肉品中的沙门氏菌Real-time PCR...应用SYBR Green I染料能选择性结合双链DNA的特点,可检测到沙门氏菌fimI基因特异性靶序列扩增所产生的荧光信号,通过熔解曲线可知其熔点值约为85.6℃,而对其他非沙门氏菌则检测不到荧光信号。建立了一种肉品中的沙门氏菌Real-time PCR检测方法,用该方法检测市售牛肉、香肠中的沙门氏菌,其检测灵敏度分别为13,12 cfu/25 g,从样品的处理到得出检验结果可以在10 h内完成。该检测方法具有简便、快速、特异性强、敏感度高等特点。展开更多
目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-...目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-Ti me PCR方法有很好的特异性与敏感性,所检测沙门菌结果均为阳性,而非沙门菌均为阴性;标准曲线相关系数为R2=0.993,其敏感性为5CFU。运用该方法对108份鸡粪便、50份鸡肉以及58份水样进行检测,阳性率分别为3.7%(6/108)、4%(2/50)和3.4%(2/58),与传统细菌分离检测结果相符。结论结果表明该方法具有简便、快速、特异性强、敏感性高等特点,此研究为环境及疾病诊断中沙门菌快速检测提供了新方法。展开更多
基金supported by the National Natural Science Foundation of China [Grant agreement 31502124]the National Science and Technology Major Project of China [Grant agreement 2018ZX10733402]
文摘Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology,and screened multiple AMR genes in broiler fecal samples.Methods A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection.A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.Results The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction.The sensitivity rate,specificity rate,positive predictive value,negative predictive value and correct indices were 99.30%,98.08%,95.31%,99.79%,and 0.9755,respectively.Utilizing this assay,we demonstrate that AMR genes are widely spread,with positive detection rates ranging from 0 to 97.07%in 273 broiler fecal samples.bla CTX-M,bla TEM,mcr-1,fex A,cfr,optr A,and int I1 showed over 80%prevalence.The dissemination of AMR genes was distinct between the two farms.Conclusions We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples.The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.
基金supported by the National Natural Science Foundation of China (31872484) to Zhang Qianyithe Non-profit Key Program of Veterinary Drug Industry from China Institute of Veterinary Drug Control (GY202011) to Xia Yingju。
文摘With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 and spread quickly across the country.It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.Atypical porcine pestivirus(APPV) was first detected in Guangdong Province,China,in 2016,which mainly harms piglets and has a local epidemic situation in southern China.These three diseases have similar clinical symptoms in pig herds,which cause considerable losses to the pig industry.They are difficult to be distinguished only by clinical diagnosis.Therefore,developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.In this study,three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV(5’ UTR),African swine fever virus(ASFV)(B646L),and APPV(5’ UTR),followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.The results showed that the method did not cross-react with other swine pathogens(porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),foot-and-mouth disease virus(FMDV),pseudorabies virus(PRV),porcine parvovirus(PPV),and bovine viral diarrhea virus BVDV).The sensitivity results showed that CSFV,ASFV,and APPV could be detected as low as 1 copy μL–1;the repeatability results showed that the intra-assay and interassay coefficient of variation of ASFV,CSFV,and APPV was less than 1%.Twenty-two virus samples were detected by the multiplex real-time PCR,compared with national standard diagnostic and patented method assay for CSF(GB/T 27540–2011),ASF(GB/T 18648–2020),and APPV(CN108611442A),respectively.The sensitivity of this triple real-time PCR for CSFV,ASFV,and APPV was almost the same,and the compliance results were the same(100%).A total of 451 clinical samples were detected,and the results showed that the positive rates of CSFV,ASFV,and APPV were 0.22% (1/451),1.3%(6/451),and 0%(0/451),respectively.This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV,ASFV,and APPV.
文摘Objective To describe correlation between multiple genetic tumor markers,carcinoembryonic antigen (CEA),cytokeratin 20 (CK20),and Survivin,and clinicopathological features of colorectal cancer (CRC) and to assess prognostic diagnosis value in cancer recurrence and metastasis.Methods A total of 92 patients with CRC,68 patients with precancerous lesions,and 29 control volunteers were collected for the detection of CEA,CK20,and Survivin expressions by using quantitative Real-Time PCR technology.Associations among these measurements and clinicopathological features of CRC,and cancer recurrence and metastasis rates in 4-year follow-up were analyzed.Results No mRNA expressions of CEA,CK20,or Survivin were detected in the control group.Expressions of CEA,CK20,and Survivin were 41.3%,47.8%,and 72.8% in CRC patients,respectively.The expressions of genetic tumor markers were related to the clinical stage and lymph node metastasis.In patients with Survivin high expression,4-year survival rate was significantly lower than that in Survivin low expression.The multiple tumor markers assay for CRC patients showed higher specificity and positive detection rate than single marker assay.Patients with CEA,CK20,and Survivin simultaneous expressions had significantly higher 4-year recurrence rate and death rate than those with only one or two markers expression.ConclusionMultiple tumor markers assay including CEA,CK20,and Survivin in peripheral blood by quantitative Real-Time PCR can be an ideal method for the surveillance of the recurrence and prognosis for CRC patients.
基金Supported by Key Special Project for Breeding and Cultivation of GMO Varieties(2011ZX08001-001,2014ZX0800101B)Special Fund from the Department of Finance of Hubei Province(2011-2015)Collaborative Breeding Project for Rice(2013-2017)
文摘In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM rice. And the identification of GM rice is of great significance. Therefore, in the present study, the po- tential problems in the identification of GM rice with PCR were analyzed both at a technical level and from a theoretical perspective. In addition, PCR detection on the transgenic elements： promoter, terminator, internal reference gene and target gene was discussed, respectively. The possible solutions were proposed based on the principles of plant virology and genetic engineering.
文摘Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resistance necessitates updating diagnostic techniques to ensure higher sensitivity and specificity, especially with advancements in science and medicine. This study aimed to evaluate the prevalence of UTIs and antibiotic resistance profiles through urine culture, as well as to identify Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp. in urine samples using a molecular approach with multiplex real-time PCR. From May 3 to July 25, 2023, at the Pietro Annigoni Biomolecular Research Center (CERBA) and Saint Camille Hospital of Ouagadougou (HOSCO), 209 urine samples collected from patients with suspected UTIs were analyzed using both urine culture and multiplex real-time PCR. Among the 209 patients, 52.15% were male and 47.85% female, with an average age of 46.87 ± 21.33 years. Urine cultures revealed an overall UTI prevalence of 23.44%, with a prevalence of 8.13% in men versus 15.31% in women (P = 0.023). The bacterial prevalence rates were as follows: Escherichia coli (12.92%), Klebsiella spp. (7.18%), Enterobacter cloacae (1.44%), Staphylococcus aureus (0.96%), and other bacteria. Klebsiella spp. demonstrated 100% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, while Escherichia coli showed 96.2% and 65.4% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, respectively. PCR analysis of the target bacteria revealed mono-infection prevalence rates of Klebsiella pneumoniae (10.39%), Klebsiella oxytoca (7.79%), and Acinetobacter spp. (7.79%), along with a co-infection prevalence rate of Klebsiella pneumoniae/Acinetobacter spp. (1.30%). This study demonstrated that PCR, with its high sensitivity and specificity, could effectively distinguish Klebsiella pneumoniae from Klebsiella oxytoca and detect Acinetobacter spp. in less than 24 hours—something urine culture alone could not achieve. The relative ease of automating urine PCR testing, combined with its diagnostic accuracy and rapid turnaround time, makes it a valuable addition to modern medical practice for the laboratory diagnosis of UTIs.
文摘目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-Ti me PCR方法有很好的特异性与敏感性,所检测沙门菌结果均为阳性,而非沙门菌均为阴性;标准曲线相关系数为R2=0.993,其敏感性为5CFU。运用该方法对108份鸡粪便、50份鸡肉以及58份水样进行检测,阳性率分别为3.7%(6/108)、4%(2/50)和3.4%(2/58),与传统细菌分离检测结果相符。结论结果表明该方法具有简便、快速、特异性强、敏感性高等特点,此研究为环境及疾病诊断中沙门菌快速检测提供了新方法。
