Immunocapture UPPCR combined with DGGE for rapid detection of Shigella species

AIMS: To develop an immunocapture universal primer PCR (iUPPCR) combined with denaturing gradient gel electrophoresis (DGGE) and evaluate it as a method permitting rapid detection of Shigella species and their serotypes. METHODS AND RESULTS: This method amplifying the conserved regions of bacterial 16S rRNA genes of different species or serotypes of Shigella dysentery bacilli captured and enriched by polyvalent antibodies can detect and distinguish causative pathogens rapidly. Four serotypes from three Shigella species including Shigella dysenteriae serotype 1, Shigella boydii serotype 1, Shigella flexneri serotypes 1a and 3a were examined. CONCLUSION: Our approach could be adopted for not only axenic bacterial population but also mixed communities and achieve rapid detection of various bacteria from the same genus or species in one sample. SIGNIFICANCE AND IMPACT OF THE STUDY: The iUPPCR-DGGE method was shown to be more convenient than serotype-specific-antibody-based method of iUPPCR for Shigella species detection and it could be also applied to the quick detection for other kinds of pathogens with many serotypes.     

Specificity of the serological diagnosis of dysentery using erythrocytic reagents

The serological specificity of the diagnosis of dysentery, made by different methods with the use of antigenic and antibody erythrocyte diagnostic agents under the conditions of the circulation of different Shigella species and subspecies in a given locality, has been compared. The method for the determination of the diagnostic titer of the total serum antibody activity has proved to be the least specific. The methods for the detection of Shigella antigens, especially over time, in patients' excretions and for the determination of serum antibody activity show complete specificity. The combination of specificity and sensitivity makes the detection of Shigella antigens in feces by means of the passive hemagglutination test and the antibody neutralization test the method of choice for the diagnosis of dysentery.     

Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid

A method was developed for the detection of the fecal coliform bacterium Escherichia coli, using the polymerase chain reaction and gene probes, based on amplifying regions of the uid gene that code for beta-glucuronidase, expression of which forms the basis for fecal coliform detection by the commercially available Colilert method. Amplification and gene probe detection of four different regions of uid specifically detected E. coli and Shigella species, including beta-glucuronidase-negative strains of E. coli; no amplification was observed for other coliform and nonenteric bacteria.     

Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid.

A method was developed for the detection of the fecal coliform bacterium Escherichia coli, using the polymerase chain reaction and gene probes, based on amplifying regions of the uid gene that code for beta-glucuronidase, expression of which forms the basis for fecal coliform detection by the commercially available Colilert method. Amplification and gene probe detection of four different regions of uid specifically detected E. coli and Shigella species, including beta-glucuronidase-negative strains of E. coli; no amplification was observed for other coliform and nonenteric bacteria.     

Detecting low concentrations of Shigella sonnei in environmental water samples by PCR

Outbreaks of Shigella sonnei associated with contaminated water have been reported and methods for the simultaneous detection of Shigellae and enteroinvasive Escherichia coli in water samples have been developed with detection limits of 10(1)-10(2) CFU mL(-1) of water. Because 10(1)-10(2)Shigellae can cause disease, a more sensitive detection method as an addition to the existing methods for detection of Shigella sonnei in water samples is reported here. Initially, 33 Shigella sonnei and 72 non-Shigella sonnei isolates were tested and one primer pair was found capable of specifically amplifying a 369-bp insertion sequence 1 (IS1) fragment from all 33 Shigella sonnei isolates and one Shigella dysenteriae ATCC isolate by PCR. The detection method was developed, which included filtration of 50 mL of water through a membrane and application of PCR to the membrane using this primer pair. Environmental water samples with total bacterial numbers of 384-2.84 x 10(7) CFU L(-1) were collected and seeded with 13 Shigella sonnei and the Shigella dysenteriae ATCC isolates. Detection limits were determined as 1.7-24.7 and 270-8000 CFU per 50 mL of water, respectively, using this detection method.     

多功能悬浮点阵技术快速检测肠道致病菌的初步应用研究

目的建立以多功能悬浮点阵技术为基础的临床常见肠道致病菌的快速检测方法。方法以细菌16S rDNA基因保守区序列设计1对通用引物,采用不对称PCR扩增7种临床常见肠道致病菌标准菌株,多功能悬浮点阵技术对不同菌株的PCR产物进行检测以验证相应菌种探针的特异性,最后对48份粪便标本进行肠道致病菌高通量快速检测。结果 7种临床常见肠道致病标准菌株的不对称PCR得到了大量单链产物,其产物用多功能悬浮点阵技术的检测特异性为100%,48份粪便标本不对称PCR产物可与相应探针发生特异性结合,且在多功能悬浮点阵技术的相应检测信号大于阴性对照3倍以上,5种细菌的多功能悬浮点阵技术检测结果与培养鉴定结果符合率100%,48份标本PCR产物均与志贺菌属探针发生杂交反应(阳性率100%)。结论 16S rDNA可以作为细菌快速鉴定的靶序列,不对称PCR产物可以显著提高与悬浮芯片杂交检测的灵敏度,多功能悬浮点阵技术在鉴定细菌方面具有简单快速、高通量、高检出率等特点,可以作为细菌快速鉴定的一种新方法,但无法鉴别志贺菌属和大肠埃希菌属。     

环介导等温扩增技术在食品中痢疾志贺氏菌快速检测

【目的】探讨、优化基于环介导等温扩增技术(LAMP)快速检测常规食品中感染性痢疾志贺氏菌的方法。【方法】在NCBI数据库中搜索获取志贺氏菌的特异性基因高度保守区,设计3对LAMP反应引物,建立、优化该LAMP可视化检测方法,并评价其特异性、灵敏度,同时与PCR检测方法和传统检测方法对比,进行结果统计分析。【结果】5株志贺氏菌标准菌株样品均检测为阳性,11株非志贺氏菌标准菌株样品均检测为阴性,无交叉反应。最低检验限为1.6×101 CFU/反应(或1.6×101 CFU/m L),且经比较,LAMP检测灵敏度比PCR检测高出1个数量级。通过对161份实际样品和人工污染样品进行检测,LAMP检测与传统方法检测结果具有较高的一致性。【结论】LAMP具有检测过程快速简便、检测结果稳定、可靠的特点,适用于对常规食品中感染性痢疾志贺氏菌的高效、快速检测需求。     

Improved procedure of selective enrichment of Shigella in the presence of Escherichia coli by use of 4-chloro-2-cyclopentylphenyl beta-D-galactopyranoside.

A previously described procedure for the selective enrichmen of Shigella in competition with E. coli has been modified and tested with a total of 48 strains of the four Shigella species. The new enrichment medium consists of 1,5-strength trypticase soy broth, 1 mM 4-chloro-2-cyclopentylphenyl beta-D-galactopyranoside (CPPG), 0.25% lactose, and 0.1 M citrate buffer (pH 6.2). In competition with a 1000-fold higher population of E. coli than Shigella, 42 of 48 strains from the four species of Shigella were selectively enriched by the new method. Different lots of CPPG did not appear to affect the performance of the medium.     

Specific detection of Salmonella spp. by multiplex polymerase chain reaction.

Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. phoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-1i primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonella species and are not present in Shigella, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of Salmonella species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 10(2) CFU after 25 cycles of PCR and 1 (10(0)) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as Salmonella species by the use of conventional cultural techniques. In addition, positive amplifications resulted from Salmonella species in environmental samples including soil and water.     

Simultaneous detection of six human diarrheal pathogens by using DNA microarray combined with tyramide signal amplification

Multiplex PCR and DNA microarray were combined with tyramide signal amplification (TSA) to develop a reliable method suitable for simultaneous detection of six species of human diarrheal pathogens (Yersinia enterocolitica, Shigella spp, Salmonella typhi, Brucella spp, Vibrio cholera and Escherichia coli O157:H7). Meanwhile, our method could distinguish V. cholera serotype O1 from O139, and O157:H7 from O157: non-H7. This assay conferred a specificity of 100% for target pathogens. The limit of detection was 103 degrees CFU/mL approximately. The results of 98.6% (357/362) clinical specimens and 100% (5/5) mocked double-blind samples were the same to that from conventional assay. Consequently this assay is sensitive and a specific tool suitable for diagnostic detection and surveillance of multiple human pathogens.     

Results of testing a new method of using antibiotics in nutrient media for the isolation of Shigella: the 2-streak method]

A new variant of media with antibiotics for isolation of Shigella, i.e. a method of 2 streaks each containing different antibiotics was tested in analysis of excrements from patients with acute dysentery. It was found that the new method is more effective than the well known method of gradient plates (isolation of Shigella in one series of the experiments amounted to 85.2 and 64.7 per cent respectively, and in the other series of the experiments the respective figures were 95.4 and 89.3 per cent). Its efficiency was lower as compared to the procedure of inoculation onto 2 plates, i.e. onto the media with and without an antibiotic (isolation of Shigella was 67.5 and 77.4 per cent respectively). The new method provided a higher frequency of Shigella isolation as compared to inoculation onto the media without an antibiotic, as well as onto any of the media used with one antibiotic. The method of 2 streaks offers wider possibilities for choosing the antibiotics for adding to the nutrient medium, as well as estimation of the antibioticograms and species structures of Shigella distributed in a concrete area.     

Electrical resistance of bacterial culture suspension as indicator of epidemic and sporadic variants of Shigella

For the first time method of measurement of electric resistance of bacterial suspension was used for differentiation of "epidemic" and "sporadic" (further used without quotes) variants of Shigella species. Study of 40 strains of S. flexneri and S. sonnei revealed significant differences in electric resistance of cultures isolated during epidemic outbreaks after the growth during 1 or 6 hours - 18.1+/-8.3 kOhm and 84.6+/-7.8 kOhm respectively (increase in 4.9-fold; p<0.01), while non significant differences were observed in cultures isolated from sporadic cases after the growth during 1 or 6 hours - 21.1+/-9.3 and 57.1+/-11.3 kOhm respectively (increase in 2.5-fold; p>0.05). Express-method for differentiation of epidemic and sporadic variants of Shigella can be useful for timely detection of outbreaks beginning (preoutbreak).     

The possibility of using the demonstration of Shigella antigens in the study of the epidemic process in dysentery

The detection of Shigella antigens in feces in accordance with the results of the passive hemagglutination test is more effective than the bacteriological method for finding out persons infected with Shigella, especially in the absence of pronounced clinical manifestations of dysentery, and can be used in the study of the mechanism of the epidemic process. The examination of two groups, each consisting of permanent and temporary members, has revealed that the use of this method for detection of the antigen makes it possible to perform the epidemiological analysis of morbidity and, in particular, to determine the source(s) of infection.     

Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels

Multiplex PCR amplification of invA and virA genes was developed enabling simultaneous detection in mussels of Salmonella spp. and Shigella spp., respectively. Simultaneous amplification of products of 215 and 275 bp was obtained either by using mixtures of individual strains of Sh. dysenteriae and Salm. typhimurium or spiked contaminated mussels with both bacteria. In the case of the mussels, 10-100 cells of Salmonella spp. and Shigella per millilitre of homogenate were detected by the multiplex PCR following a pre-enrichment step to increase sensitivity and to ensure that detection was based on the presence of cultivable bacteria. Also, the sensitivity and specificity of this method was evaluated. Multiplex PCR amplification was shown to be an effective, sensitive and rapid method for the simultaneous detection of pathogens in mussels.     

环介导等温扩增联合横向流动试纸条可视化检测志贺氏菌

【目的】将环介导等温扩增技术(LAMP)与横向流动试纸条(LFD)联合应用,建立一种可应用于志贺氏菌快速检测的LAMP-LFD技术。【方法】以福氏志贺氏菌的侵袭性质粒抗原H(ipa H)基因为检测靶标设计3对特异性引物(其中上游内引物Sfl-ipa H-FIP由生物素标记),进行LAMP反应;同时设计1条异硫氰酸荧光素(FITC)标记的探针Sfl-ipa H-HP,与获得的LAMP产物进行特异性杂交,杂交产物经LFD完成检测。【结果】优化后的LAMP反应条件为63°C 40 min,加上LFD结果判读共需50 min。LAMP-LFD方法能够特异性检测出福氏志贺氏菌,而对肠炎沙门氏菌等其它4种导致腹泻的致病菌和创伤弧菌等5种常见食物源性致病菌,以及4株不同大肠杆菌的检测结果呈阴性。该方法针对福氏志贺氏菌的检测灵敏度为1.0×10~2 CFU/m L或4 CFU/反应,针对人工污染鲤鱼肠组织的检测灵敏度是5.0×10~2 CFU/m L,是以LAMP外引物Sfl-ipa H-F3/Sfl-ipa H-B3的常规PCR方法的100倍。【结论】建立的LAMP-LFD技术具有操作简单、检测快速准确、检测成本低等优点,有望在志贺氏菌的常规监测和即时检测中被普及使用。     

A novel marker for the species-specific detection and quantitation of Shigella sonnei by targeting a methylase gene

Shigella sonnei is a causal agent of fever, nausea, stomach cramps, vomiting, and diarrheal disease. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the specific detection of S. sonnei using a primer pair based on the methylase gene for the amplification of a 325 bp DNA fragment. The qPCR primer set for the accurate diagnosis of Shigella sonnei was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management.     

Development of immunoreagents for the diagnostics of intestinal yersiniosis by antigen-binding lymphocytes

To diagnose intestinal yersiniosis, the detection of antigen-binding lymphocytes (ABL) with respect to Y. enterocolitica O3, O5 and O9 is proposed. Experiments on the immunization of rabbits with Y. enterocolitica LPS of these serovars revealed that immunoreagents, according to the data of cross antigen-dependent rosette formation and its inhibition, had species specificity (ABL of rabbits immunized with Y. enterocolitica did not interact with Salmonella, Shigella and Y. pseudotuberculosis antigens) and serovar specificity. Cross reactions in the detection of ABL by means of specific Y. enterocolitica O9 and Brucella melitensis immunoreagents in rabbits immunized with Y. enterocolitica were absent during the first days and could be detected only of day 25 after the injection of the immunogen. The method for the detection of ABL with the use of newly developed reagents could be used in clinics for the diagnostics of intestinal yersiniosis.     

Detection of coliform bacteria in water by polymerase chain reaction and gene probes

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.     

Detection of coliform bacteria in water by polymerase chain reaction and gene probes.

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.