[Objective] To establish a Taqman real-time PCR for detection of Salmonella in pet food. [Method] A pair of primers and a probe were designed based on published nucleotide sequence of invA gene encoding the invasion p...[Objective] To establish a Taqman real-time PCR for detection of Salmonella in pet food. [Method] A pair of primers and a probe were designed based on published nucleotide sequence of invA gene encoding the invasion protein of Salmonella enterica. [Result] The assay detects Salmonella specifically. The detection limit of the real-time PCR was 17 CFU/test (25 μL/test) for the positive strain. This method was effective to detect artificially contaminated pet food. [Conclusion] The results showed that Taqman PCR assay was rapid and accurate for detection of Salmonella from infected pet food.展开更多
Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three t...Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.展开更多
Rapid detection of target foodborne pathogens plays more and more significant roles in food safety,which requires the efficiency,sensitivity,and accuracy.In this research,we proposed a new st rategy of isothermal-mole...Rapid detection of target foodborne pathogens plays more and more significant roles in food safety,which requires the efficiency,sensitivity,and accuracy.In this research,we proposed a new st rategy of isothermal-molecular-amplification integrated with lateral-flow-strip for rapid detection of Salmonella without traditional enrichment-culture.Th e designed syringe-assisted-filtration can contribute to simultaneous collection and concentration of target bacterium from vegetable samples in just 3 min,resolving the drawbacks of traditional random sampling protocols.After simple and convenient ultrasonication,samples can be directly amplified at 39℃ in 25 min and the amplicons are qualitatively and quantitatively analyzed with the designed lateral-flow-strip in 5 min.Finally,satisfied results have been achieved within 40 min,which greatly improve the efficiency while the accuracy is also guaranteed.Furthermore,all detection steps can be completed under instrument-free conditions.This method will hold great promise for target pathogen detection in the resource-limited district,or for emergency on-site identification.展开更多
Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in dise...Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in disease management.In this study,we adapted a quantitative real-time PCR(qPCR)assay to droplet digital PCR(ddPCR)format for A.citrulli detection by optimizing reaction conditions.The performance of ddPCR in detecting A.citrulli pure culture,DNA,infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR,qPCR,and dilution plating method.The lowest concentrations detected(LCD)by ddPCR reached up to 2 fg DNA,and 102 CFU mL–1 bacterial cells,which were ten times more sensitive than those of the qPCR.When testing artificially infested watermelon and melon seed,0.1%infestation level was detectable using ddPCR and dilution plating method.The 26 positive samples were identified in 201 commercial seed samples through ddPCR,which was the highest positive number among all the methods.High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.展开更多
<span><i><span style="font-family:""><i></span></i></span><span><span><i><span style="font-family:"">Salmonella enterica&...<span><i><span style="font-family:""><i></span></i></span><span><span><i><span style="font-family:"">Salmonella enterica</span></i></span></span><span><span><i><span style="font-family:""></i></span></i></span></span><span><span><span style="font-family:""> serovars is a leading cause of human gastroenteritis, and the incidence of salmonellosis is constantly increasing, causing millions of infections and many deaths annually. The detection of the pathogen in optimal terms is an essential factor for reducing the impact on the human body. In this work</span></span></span><span><span><span style="font-family:"">,</span></span></span><span><span><span style="font-family:""> SYBR Green I-based qPCR method of detection and quantifica<span>tion of </span></span></span></span><span><span><span style="font-family:""><i></span></span></span><span><span><i><span style="font-family:"">Salmonella enterica</span></i></span></span><span><span><i><span style="font-family:""></i></span></i></span></span><span><span><i><span style="font-family:""> </span></i></span></span><span><span><span style="font-family:"">was</span></span></span><span><span><span style="font-family:""> developed and validated. For detection o</span></span></span><span><span><span style="font-family:"">f </span></span></span><span><span><span style="font-family:""><i></span></span></span><span><span><i><span style="font-family:"">Salmonella enterica</span></i></span></span><span><span><i><span style="font-family:""></i></span></i></span></span><span><span><span style="font-family:""> subsp.<i> </i></span></span></span><span><span><i><span style="font-family:""><i></span></i></span></span><span><span><i><span style="font-family:"">enterica</span></i></span></span><span><span><i><span style="font-family:""></i></span></i></span></span><span><span><span style="font-family:"">, two pairs of primers were designed using publically available Primer-BLAST software. Primer efficiency was calculated <span>by establishing a standard curve. The specificity, sensitivity, accuracy, and</span> precision of PCR results were tested. Both primer pairs showed an acceptable performance, proving the developed techniques were sensitive, reliable and precise. The validated <span>qPCR technology has a good potential to replace the traditional culture method in microbial diagnosis.展开更多
A real-time RT-PCR (RT-qPCR) assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequ...A real-time RT-PCR (RT-qPCR) assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.展开更多
Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed t...Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.展开更多
Venereal diseases are considered to be the most prevalent infectious diseases in the worldwide. China is now faced with a year-by-year increasing incidence of sexually transmitted diseases (STD), which are spreading...Venereal diseases are considered to be the most prevalent infectious diseases in the worldwide. China is now faced with a year-by-year increasing incidence of sexually transmitted diseases (STD), which are spreading from high-risk groups to the general population. Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum and herpes simplex virus-2 (HSV-2) are always regarded as the most common venereal pathogens. The "golden standard" for testing Neisseria gonorrhoeae remains to be bacteria culture or microscopic examination.展开更多
A set of universal loop-mediated isothermal amplification (LAMP) primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato (B. burgdorferi s.I.) in human samples. The sensitivity of LAM...A set of universal loop-mediated isothermal amplification (LAMP) primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato (B. burgdorferi s.I.) in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.展开更多
We developed one species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Verticillium albo-atrum in diseased plant tissues and soil. Based on differences in internal transcribed spacer (...We developed one species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Verticillium albo-atrum in diseased plant tissues and soil. Based on differences in internal transcribed spacer (ITS) sequences of Verticillium spp., a pair of species-specific primers, Vaal/Vaa2, was synthesized. After screening 17 isolates of V. alboatrum, 121 isolates from the Ascomycota, B asidiomycota, Deuteromycota, and Oomycota, the Vaal/Vaa2 primers amplified only a single PCR band of approximately 330 bp from V. albo-atrum. The detection sensitivity with primers Vaal/Vaa2 was 10 fg of genomic DNA. Using ITS1/ITS4 as the first-round primers, combined with Vaa1/Vaa2, the nested PCR procedures were developed, and the detection sensitivity increased 1 000-fold to 10 ag. The detection sensitivity for the soil pathogens was 100-conidiag^-1 soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.展开更多
Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a ma...Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a major oyster production area in Southwestern China. Methods Oyster samples were collected monthly from farms, markets, and restaurants, from January to December 2016. Norovirus was detected and quantified by one-step reverse transcription-droplet digital polymerase chain reaction(RT-ddPCR). Results A total of 480 oyster samples were collected and tested for norovirus genogroups I and II. Norovirus was detected in 20.7% of samples, with genogroup II predominating. No significant difference was observed in norovirus prevalence among different sampling sites. The norovirus levels varied widely, with a geometric mean of 19,300 copies/g in digestive glands. Both norovirus prevalence and viral loads showed obvious seasonality, with a strong winter bias. Conclusion This study provides a systematic analysis of norovirus contamination ‘from the farm to the fork' in Guangxi. RT-ddPCR can be a useful tool for detection and quantification of low amounts of norovirus in the presence of inhibitors found particularly in foodstuffs. This approach will contribute to the development of strategies for controlling and reducing the risk of human illness resulting from shellfish consumption.展开更多
Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry.Therefore,a rapid,reproducible,and sensitive method for detection and quantification of this pathogen is needed urgent...Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry.Therefore,a rapid,reproducible,and sensitive method for detection and quantification of this pathogen is needed urgently.To achieve this purpose,we developed a TaqMan-based real-time PCR assay for detection and quantification of E.tarda.The assay targets the hemolysin activator HlyB domain protein of E.tarda.Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction.A standard curve was generated from the threshold cycle values(y) against log10(E.tarda genomic DNA concentration) as x.The intra-and inter-assay coefficient of variation(CV) values were less than 2.06% and 1.05% respectively,indicating that the assay had good reproducibility.This method is highly specific to E.tarda strains,as it shows no cross-reactivity to Edwardsiella ictaluri,a member of the same genus,or to nine other fish-pathogenic bacteria species belonging to three other genera.This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E.tarda in clinical samples.展开更多
Meloidogyne vitis is a new root-knot nematode parasitic on grape root in Yunnan Province,China.In order to establish a rapid,reliable and specific molecular detection method for M.vitis,the species-specific primers we...Meloidogyne vitis is a new root-knot nematode parasitic on grape root in Yunnan Province,China.In order to establish a rapid,reliable and specific molecular detection method for M.vitis,the species-specific primers were designed with rDNA-ITS(ribosomal DNA internal transcribed spacer)gene fragment as the target.The reaction system was optimized and the reliability,specificity and sensitivity of primer were testified,therefore,a rapid PCR detection method for M.vitis was established.The result showed that the optimal annealing temperature of the primers was 53℃,which was suitable for the detection of different life stages of M.vitis.Specificity test showed that the specific fragment size of 174 bp was obtained from M.vitis,but other five non-target nematodes did not have any amplification bands,thus effectively distinguish M.vitis and the other five species,and could specifically detect the M.vitis from mixed populations.Sensitivity test showed that this PCR technique could detect the DNA of a single second-stage juvenile(J_(2))and 10^(-4)female.Futhermore,this PCR technique could be used to detect directly M.vitis from soil samples.The rapid,sensitive and specific PCR molecular detection technique could be used for the direct identification of a single J_(2)of M.vitis and the detection of M.vitis in mixed nematode populations and the detection of two J_(2)s or one male in 0.5 g soil samples,which will provide technical support for the investigation of the occurrence and damage of M.vitis and the formulation of efficient green co ntrol strategies.展开更多
Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitativ...Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitative fluorescence signal collection process was set in the extension stage.Results:Three-step amplification has the advantages of wide application range;improved accuracy;and reduced primer design requirements.Conclusion:The interference of non-specific amplification signals was effectively avoided,the melting curve plotting process was omitted,the reaction time was shortened,and the detection accuracy was improved.展开更多
Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide.Genome structure of influenza D virus(IDV)is identical to that of influenza C virus(I...Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide.Genome structure of influenza D virus(IDV)is identical to that of influenza C virus(ICV),and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology.To date,the prevalence of ICV and IDV in China is unclear,but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential.To efficiently monitor ICV and IDV,it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research.A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed.TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed.This method exhibited good specificity and sensitivity,and the detection limit reached 1 × 10^(1) copies/pL of plasmid standards of each pathogen.Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method.One positive sample of IDV was detected,and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing.The duplex real-time PCR detection method represents a sensitive and specific tool to detect IG/and IDV,It provides technical support for virus research and clinical diagnosis of ICV and IDV.This information will benefit animal and human health.展开更多
Objective:To study virulence and regulatory genes(hlyA,ctxB,tcpI) in clinical strains of Vibrio ckolerae(V.cholerae),simultaneously.Methods:Three important genes,tepI,hlyA and ctxB were used for detection of toxigenic...Objective:To study virulence and regulatory genes(hlyA,ctxB,tcpI) in clinical strains of Vibrio ckolerae(V.cholerae),simultaneously.Methods:Three important genes,tepI,hlyA and ctxB were used for detection of toxigenic and pathogenic V.cholera by chain reaction assay method. Results:According to the results of the PCR,the incidence of hlyA,tcpI,and ctxB genes in clinical isolates was obtained as 94.7%(72 sample),90.8%(69 sample),and 92.1%(70 sample), respectively.Five strains possessed all genes except ctxB,six strains possessed all genes except tcpI,four strains possessed all genes except hlyA,one strain possessed only hlyA and 60 strains contained a combination of three genes.Including hlyA,ctxB and tcpI,Conclusions:Result show that this method could be reliable to detect toxigenic-pathogenic strains of V.cholerae in Iran.展开更多
Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In t...Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQ- PCR assay was determined to be as low as five copies of the target sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values (y) against the common logarithmic copies (logl0n~ as x; n~ is copy number) of pGEM-16S rDNA was generated. The results of intra- and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.展开更多
Objective To develop an in situ PCR in combination with flow cytometry (ISPCR-FCM) for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibri...Objective To develop an in situ PCR in combination with flow cytometry (ISPCR-FCM) for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene (ctxAB gene). Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low (10^5 cells/mL), while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.展开更多
To develop a nested PCR assay for the detection of cattle-derived Cyclospora sp., two pairs of primers were designed on the basis of the cattle-derived Cyclospora sp. sequences. These primers selectively amplified a 1...To develop a nested PCR assay for the detection of cattle-derived Cyclospora sp., two pairs of primers were designed on the basis of the cattle-derived Cyclospora sp. sequences. These primers selectively amplified a 168-bp DNA fragment of the 18S rRNA gene of cattle-derived Cyclospora sp. With these primers, a nested PCR assay for the detection of cattlederived Cyclospora sp. was developed. The nested PCR assay was specific and there is no cross-reaction with other parasites, such as Eimeria spp., Cryptosporidium spp., Giardia sp., Toxoplasma sp., Trichuris sp. and cattle ciliate. The assay was able to detect as low as 2.85 × 10^-2 fg of the control positive DNA. The results of the detection of clinical samples indicated that the assay coincided with microscopic examination. The results show that the nested PCR assay will be an effective tool for the detection of Cyclospora sp. in cattle feces.展开更多
AIM: To analyze the difference of intestinal microbial community diversity between healthy and (S. enteritidis) orally infected ducklings.METHODS: Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR was applied...AIM: To analyze the difference of intestinal microbial community diversity between healthy and (S. enteritidis) orally infected ducklings.METHODS: Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR was applied to analyze the intestinal microbial community diversity and dynamic change including duodenum, jejunum, ileum, cecum and rectum from healthy ducklings and 7-day-old ducklings after oral infection with S. enteritidis at different time points.RESULTS: The intestinal microbial community of the control healthy ducklings was steady and the ERIC-PCR band numbers of the control healthy ducklings were the least with rectum and were the most with caecum. ERIC-PCR bands of orally inoculated ducklings did not obviously change until 24 h after inoculation (p.i.). The numbers of the ERIC-PCR bands gradually decreased from 24 h to 72 h p.i., and then, with the development of disease, the band numbers gradually increased until 6 d p.i. The prominent bacteria changed because of S. enteritidis infection and the DNAstar of staple of ERIC-PCR showed that aerobe and facultative aerobe (Escherichia coli, Shigella, Salmonella) became preponderant bacilli in the intestine of orally infected ducklings with SE.CONCLUSION: This study has provided significant data to clarify the intestinal microbial community diversity and dynamic change of healthy and S. enteritidis orally infected ducklings, and valuable insight into the pathogenesis of S. enteritidis infection in both human and animals.展开更多
基金Supported by the Project of General Administration of Quality Supervision,Inspection and Quarantine of the People's Republic of China( 201110034)
文摘[Objective] To establish a Taqman real-time PCR for detection of Salmonella in pet food. [Method] A pair of primers and a probe were designed based on published nucleotide sequence of invA gene encoding the invasion protein of Salmonella enterica. [Result] The assay detects Salmonella specifically. The detection limit of the real-time PCR was 17 CFU/test (25 μL/test) for the positive strain. This method was effective to detect artificially contaminated pet food. [Conclusion] The results showed that Taqman PCR assay was rapid and accurate for detection of Salmonella from infected pet food.
基金funded by the National Key R&D Program of China[2021YFC2301102]National Natural Science Foundation of China[82202593]Key R&D Program of Hebei Province[223777100D].
文摘Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.
基金financially supported by the grants of the NSFC(32172295,21804028)the key R&D program of Anhui(201904d07020016)+5 种基金the Anhui Provincial NSF(1908085QC121)the Fundamental Research Fund for central university(JZ2019HGTB0068)the China Postdoctoral Science Foundation(2019M652167)the Fund of State Key Lab of Chemo/Biosensing and Chemometrics(Hunan University),the postdoc grant of Anhui(2020B412)Young and Middle-aged Leading Scientists,Engineers and Innovators of the XPCC(2019CB017)China Agriculture Research System-48(CARS-48).
文摘Rapid detection of target foodborne pathogens plays more and more significant roles in food safety,which requires the efficiency,sensitivity,and accuracy.In this research,we proposed a new st rategy of isothermal-molecular-amplification integrated with lateral-flow-strip for rapid detection of Salmonella without traditional enrichment-culture.Th e designed syringe-assisted-filtration can contribute to simultaneous collection and concentration of target bacterium from vegetable samples in just 3 min,resolving the drawbacks of traditional random sampling protocols.After simple and convenient ultrasonication,samples can be directly amplified at 39℃ in 25 min and the amplicons are qualitatively and quantitatively analyzed with the designed lateral-flow-strip in 5 min.Finally,satisfied results have been achieved within 40 min,which greatly improve the efficiency while the accuracy is also guaranteed.Furthermore,all detection steps can be completed under instrument-free conditions.This method will hold great promise for target pathogen detection in the resource-limited district,or for emergency on-site identification.
基金supported by the the National Key Research and Development Program of China (2017YFD0201602)the National Natural Science Foundation of China (31401704)the Beijing Academy of Agriculture and Forestry Foundation, China (KJCX20180203)
文摘Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in disease management.In this study,we adapted a quantitative real-time PCR(qPCR)assay to droplet digital PCR(ddPCR)format for A.citrulli detection by optimizing reaction conditions.The performance of ddPCR in detecting A.citrulli pure culture,DNA,infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR,qPCR,and dilution plating method.The lowest concentrations detected(LCD)by ddPCR reached up to 2 fg DNA,and 102 CFU mL–1 bacterial cells,which were ten times more sensitive than those of the qPCR.When testing artificially infested watermelon and melon seed,0.1%infestation level was detectable using ddPCR and dilution plating method.The 26 positive samples were identified in 201 commercial seed samples through ddPCR,which was the highest positive number among all the methods.High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.
文摘<span><i><span style="font-family:""><i></span></i></span><span><span><i><span style="font-family:"">Salmonella enterica</span></i></span></span><span><span><i><span style="font-family:""></i></span></i></span></span><span><span><span style="font-family:""> serovars is a leading cause of human gastroenteritis, and the incidence of salmonellosis is constantly increasing, causing millions of infections and many deaths annually. The detection of the pathogen in optimal terms is an essential factor for reducing the impact on the human body. In this work</span></span></span><span><span><span style="font-family:"">,</span></span></span><span><span><span style="font-family:""> SYBR Green I-based qPCR method of detection and quantifica<span>tion of </span></span></span></span><span><span><span style="font-family:""><i></span></span></span><span><span><i><span style="font-family:"">Salmonella enterica</span></i></span></span><span><span><i><span style="font-family:""></i></span></i></span></span><span><span><i><span style="font-family:""> </span></i></span></span><span><span><span style="font-family:"">was</span></span></span><span><span><span style="font-family:""> developed and validated. For detection o</span></span></span><span><span><span style="font-family:"">f </span></span></span><span><span><span style="font-family:""><i></span></span></span><span><span><i><span style="font-family:"">Salmonella enterica</span></i></span></span><span><span><i><span style="font-family:""></i></span></i></span></span><span><span><span style="font-family:""> subsp.<i> </i></span></span></span><span><span><i><span style="font-family:""><i></span></i></span></span><span><span><i><span style="font-family:"">enterica</span></i></span></span><span><span><i><span style="font-family:""></i></span></i></span></span><span><span><span style="font-family:"">, two pairs of primers were designed using publically available Primer-BLAST software. Primer efficiency was calculated <span>by establishing a standard curve. The specificity, sensitivity, accuracy, and</span> precision of PCR results were tested. Both primer pairs showed an acceptable performance, proving the developed techniques were sensitive, reliable and precise. The validated <span>qPCR technology has a good potential to replace the traditional culture method in microbial diagnosis.
基金supported by the National Science and Technology Major Project of the Ministry of Science and Technology of China(No.2013ZX10004-101)
文摘A real-time RT-PCR (RT-qPCR) assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.
基金supported by grants from the Development Grant of State Key Laboratory of Infectious Disease Prevention and Control(2012SKLID204,2015SKLID505)the Ministry of Science and Technology of People’s Republic of China(No.2013ZX10004101)
文摘Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.
基金supported by Zhejiang Provincial Population and Family Planning Foundation of China (N20100011)
文摘Venereal diseases are considered to be the most prevalent infectious diseases in the worldwide. China is now faced with a year-by-year increasing incidence of sexually transmitted diseases (STD), which are spreading from high-risk groups to the general population. Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum and herpes simplex virus-2 (HSV-2) are always regarded as the most common venereal pathogens. The "golden standard" for testing Neisseria gonorrhoeae remains to be bacteria culture or microscopic examination.
基金funded by the National Key Science and Technology Projects of China(2012ZX10004219 and 2013ZX10004001)
文摘A set of universal loop-mediated isothermal amplification (LAMP) primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato (B. burgdorferi s.I.) in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.
基金We thank Dr Chen Qinghe,Dr Ko Wenhong,Dr Ho Hanhin,Dr Hu Baishi,Dr Peng Jinghuo and China General Microbiological Culture Collection Center(CGMCC)for providing some isolates.This work was supported by the China National 863 Program(2003AA249020).
文摘We developed one species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Verticillium albo-atrum in diseased plant tissues and soil. Based on differences in internal transcribed spacer (ITS) sequences of Verticillium spp., a pair of species-specific primers, Vaal/Vaa2, was synthesized. After screening 17 isolates of V. alboatrum, 121 isolates from the Ascomycota, B asidiomycota, Deuteromycota, and Oomycota, the Vaal/Vaa2 primers amplified only a single PCR band of approximately 330 bp from V. albo-atrum. The detection sensitivity with primers Vaal/Vaa2 was 10 fg of genomic DNA. Using ITS1/ITS4 as the first-round primers, combined with Vaa1/Vaa2, the nested PCR procedures were developed, and the detection sensitivity increased 1 000-fold to 10 ag. The detection sensitivity for the soil pathogens was 100-conidiag^-1 soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.
文摘Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a major oyster production area in Southwestern China. Methods Oyster samples were collected monthly from farms, markets, and restaurants, from January to December 2016. Norovirus was detected and quantified by one-step reverse transcription-droplet digital polymerase chain reaction(RT-ddPCR). Results A total of 480 oyster samples were collected and tested for norovirus genogroups I and II. Norovirus was detected in 20.7% of samples, with genogroup II predominating. No significant difference was observed in norovirus prevalence among different sampling sites. The norovirus levels varied widely, with a geometric mean of 19,300 copies/g in digestive glands. Both norovirus prevalence and viral loads showed obvious seasonality, with a strong winter bias. Conclusion This study provides a systematic analysis of norovirus contamination ‘from the farm to the fork' in Guangxi. RT-ddPCR can be a useful tool for detection and quantification of low amounts of norovirus in the presence of inhibitors found particularly in foodstuffs. This approach will contribute to the development of strategies for controlling and reducing the risk of human illness resulting from shellfish consumption.
基金Supported by the Special Fund for Agro-scientific Research in the Public Interest(No.201103034)the Construction Special Fund of Modern Agriculture and Industrial Technology Research System(No.CARS-47)
文摘Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry.Therefore,a rapid,reproducible,and sensitive method for detection and quantification of this pathogen is needed urgently.To achieve this purpose,we developed a TaqMan-based real-time PCR assay for detection and quantification of E.tarda.The assay targets the hemolysin activator HlyB domain protein of E.tarda.Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction.A standard curve was generated from the threshold cycle values(y) against log10(E.tarda genomic DNA concentration) as x.The intra-and inter-assay coefficient of variation(CV) values were less than 2.06% and 1.05% respectively,indicating that the assay had good reproducibility.This method is highly specific to E.tarda strains,as it shows no cross-reactivity to Edwardsiella ictaluri,a member of the same genus,or to nine other fish-pathogenic bacteria species belonging to three other genera.This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E.tarda in clinical samples.
基金supported by the grants from the National Key Research and Development Program of China(2018YFD0201202 and 2017YFD0200601)。
文摘Meloidogyne vitis is a new root-knot nematode parasitic on grape root in Yunnan Province,China.In order to establish a rapid,reliable and specific molecular detection method for M.vitis,the species-specific primers were designed with rDNA-ITS(ribosomal DNA internal transcribed spacer)gene fragment as the target.The reaction system was optimized and the reliability,specificity and sensitivity of primer were testified,therefore,a rapid PCR detection method for M.vitis was established.The result showed that the optimal annealing temperature of the primers was 53℃,which was suitable for the detection of different life stages of M.vitis.Specificity test showed that the specific fragment size of 174 bp was obtained from M.vitis,but other five non-target nematodes did not have any amplification bands,thus effectively distinguish M.vitis and the other five species,and could specifically detect the M.vitis from mixed populations.Sensitivity test showed that this PCR technique could detect the DNA of a single second-stage juvenile(J_(2))and 10^(-4)female.Futhermore,this PCR technique could be used to detect directly M.vitis from soil samples.The rapid,sensitive and specific PCR molecular detection technique could be used for the direct identification of a single J_(2)of M.vitis and the detection of M.vitis in mixed nematode populations and the detection of two J_(2)s or one male in 0.5 g soil samples,which will provide technical support for the investigation of the occurrence and damage of M.vitis and the formulation of efficient green co ntrol strategies.
文摘Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitative fluorescence signal collection process was set in the extension stage.Results:Three-step amplification has the advantages of wide application range;improved accuracy;and reduced primer design requirements.Conclusion:The interference of non-specific amplification signals was effectively avoided,the melting curve plotting process was omitted,the reaction time was shortened,and the detection accuracy was improved.
基金This work was financially supported by the National Key Research and Development Program of China(2017YFD0500101)the Fundamental Research Funds for the Central Universities(Y0201900459).
文摘Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide.Genome structure of influenza D virus(IDV)is identical to that of influenza C virus(ICV),and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology.To date,the prevalence of ICV and IDV in China is unclear,but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential.To efficiently monitor ICV and IDV,it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research.A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed.TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed.This method exhibited good specificity and sensitivity,and the detection limit reached 1 × 10^(1) copies/pL of plasmid standards of each pathogen.Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method.One positive sample of IDV was detected,and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing.The duplex real-time PCR detection method represents a sensitive and specific tool to detect IG/and IDV,It provides technical support for virus research and clinical diagnosis of ICV and IDV.This information will benefit animal and human health.
文摘Objective:To study virulence and regulatory genes(hlyA,ctxB,tcpI) in clinical strains of Vibrio ckolerae(V.cholerae),simultaneously.Methods:Three important genes,tepI,hlyA and ctxB were used for detection of toxigenic and pathogenic V.cholera by chain reaction assay method. Results:According to the results of the PCR,the incidence of hlyA,tcpI,and ctxB genes in clinical isolates was obtained as 94.7%(72 sample),90.8%(69 sample),and 92.1%(70 sample), respectively.Five strains possessed all genes except ctxB,six strains possessed all genes except tcpI,four strains possessed all genes except hlyA,one strain possessed only hlyA and 60 strains contained a combination of three genes.Including hlyA,ctxB and tcpI,Conclusions:Result show that this method could be reliable to detect toxigenic-pathogenic strains of V.cholerae in Iran.
基金The Special Fund for Agro-scientific Research in the Public Interest under contract No.201103034Construction Special Fund of Modern Agriculture and Industrial Technology Research System under contract No.CARS-47
文摘Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQ- PCR assay was determined to be as low as five copies of the target sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values (y) against the common logarithmic copies (logl0n~ as x; n~ is copy number) of pGEM-16S rDNA was generated. The results of intra- and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.
基金This work was supported by the Natural Sciences Foundation of China (Grant No. NSFC. 40176036).
文摘Objective To develop an in situ PCR in combination with flow cytometry (ISPCR-FCM) for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene (ctxAB gene). Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low (10^5 cells/mL), while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.
基金the National Natural Science Foundation of China (30371082, 30671577) Natural Science Foundation of Guangdong Province, China (32286).
文摘To develop a nested PCR assay for the detection of cattle-derived Cyclospora sp., two pairs of primers were designed on the basis of the cattle-derived Cyclospora sp. sequences. These primers selectively amplified a 168-bp DNA fragment of the 18S rRNA gene of cattle-derived Cyclospora sp. With these primers, a nested PCR assay for the detection of cattlederived Cyclospora sp. was developed. The nested PCR assay was specific and there is no cross-reaction with other parasites, such as Eimeria spp., Cryptosporidium spp., Giardia sp., Toxoplasma sp., Trichuris sp. and cattle ciliate. The assay was able to detect as low as 2.85 × 10^-2 fg of the control positive DNA. The results of the detection of clinical samples indicated that the assay coincided with microscopic examination. The results show that the nested PCR assay will be an effective tool for the detection of Cyclospora sp. in cattle feces.
基金The National Science &Technology Pillar Program, 2007Z06-017Program for New Century Excellent Talents in University, NCET-04-0906/NCET-06-0818+1 种基金Sichuan Province Basic Research Program, 04JY029-006-1/04JY021-100/07JY029-017Program for Key Disciplines Construction of Sichuan Province, SZD0418
文摘AIM: To analyze the difference of intestinal microbial community diversity between healthy and (S. enteritidis) orally infected ducklings.METHODS: Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR was applied to analyze the intestinal microbial community diversity and dynamic change including duodenum, jejunum, ileum, cecum and rectum from healthy ducklings and 7-day-old ducklings after oral infection with S. enteritidis at different time points.RESULTS: The intestinal microbial community of the control healthy ducklings was steady and the ERIC-PCR band numbers of the control healthy ducklings were the least with rectum and were the most with caecum. ERIC-PCR bands of orally inoculated ducklings did not obviously change until 24 h after inoculation (p.i.). The numbers of the ERIC-PCR bands gradually decreased from 24 h to 72 h p.i., and then, with the development of disease, the band numbers gradually increased until 6 d p.i. The prominent bacteria changed because of S. enteritidis infection and the DNAstar of staple of ERIC-PCR showed that aerobe and facultative aerobe (Escherichia coli, Shigella, Salmonella) became preponderant bacilli in the intestine of orally infected ducklings with SE.CONCLUSION: This study has provided significant data to clarify the intestinal microbial community diversity and dynamic change of healthy and S. enteritidis orally infected ducklings, and valuable insight into the pathogenesis of S. enteritidis infection in both human and animals.