Application and evaluation of multiple cross displacement amplification in detection of Listeria monocytogenes / 中国人兽共患病学报

Listeria monocytogenes is a significant food-borne pathogen that is capable of causing severe listeriosis in both humans and animals.Traditional techniques such as culture-based approaches have good specificity and sensitivity,while they are time-consuming and tend to be tedious.Hence,we presented a simple and rapid molecular technique using multiple cross displacement amplification (MCDA) targeting L.monocytogenes-specific gene lmo0733 for sensitive and specific detection of the pathogen.L.monocytogenes-MCDA assay used a set of ten primers spanning ten distinct regions of target sequence and was carried out at a constant temperature 61 ℃ to evaluate the specificity,sensitivity and feasibility.A total of three methods were used to confirm MCDA products,including color change of Loopamp Fluorescent Detection Reagent,real-time measurement of turbidity and agarose gel electrophoresis.The limit of detection (LoD) of the MCDA was 10 fg DNA,which was 25-fold and 250-fold more sensitive than that of the LAMP and CPA assay for detecting L.monocytogenes DNA,respectively.The detection ability of MCDA method was identical to the culture-biotechnical method for 153 rat intestinal feces samples,and was better than LAMP,CPA and conventional PCR.The results in this study indicated that the MCDA assay could be used as a rapid,sensitive and effective tool for the detection of L.monocytogenes in the food industry,medical institutions and field.

Rapid Identification and Subtyping of Enterobacter cloacae Clinical Isolates Using Peptide Mass Fingerprinting / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To establish a domestic database of Enterobacteria cloacae (E. cloacae), and improve the identification efficiency using peptide mass fingerprinting.</p><p><b>METHODS</b>Peptide mass fingerprinting was used for the identification and subtyping of E. cloacae. Eighty-seven strains, identified based on hsp60 genotyping, were used to construct and evaluate a new reference database.</p><p><b>RESULTS</b>Compared with the original reference database, the identification efficiency and accuracy of the new reference database was greatly improved at the species level. The first super reference database for E. cloacae identification was also constructed and evaluated. Based on the super reference database and the main spectra projection dendrogram, E. cloacae strains were divided into two clades.</p><p><b>CONCLUSION</b>Peptide mass fingerprinting is a powerful method to identify and subtype E. cloacae, and the use of this method will allow us to obtain more information to understand the heterogeneous organism E. cloacae.</p>

Establishment and comparison of pulsed-field gel electrophoresis, multiple-locus variable number tandem repeat analysis and automated ribotyping methods for subtyping of Citrobacter strains / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To establish and compare the pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA) and automated ribotyping for subtyping of Citrobacter strains.</p><p><b>METHODS</b>PFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system.</p><p><b>RESULTS</b>We optimized the plug preparation procedure, focused on the cell suspension concentration (turbidity of 2.5 to 3.5), SDS addition (no SDS needed) and lysis time (1 h), and selected the appropriate restriction enzyme (XbaI) and the electrophoretic parameters (1.0 s-20.0 s for 19 h) of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources (with the same species from the same place at the same time identified as the same source) and divided strains from different sources (from different years, places and hosts) into different subtypes.</p><p><b>CONCLUSION</b>PFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.</p>

Gene detection of Neisseria spp. isolated from male patients with genitourinary tract infections / 中华男科学杂志

<p><b>OBJECTIVE</b>To detect the genes of Neisseria spp. isolated from patients with male genitourinary tract infections, and to study the pathogenicity of non-gonococcal strains of Neisseria and the laboratory diagnosis for the infections caused by Neisseria spp.</p><p><b>METHODS</b>Using polymerase chain reaction and nucleotide sequencing, we amplified and sequenced 4 genes of Neisseria spp. isolated from patients with male genitourinary tract infections, including 16S rRNA, orfl, cppB and nspA.</p><p><b>RESULTS</b>Fourteen Neisseria strains were identified through analysis of the 16S rRNA gene, including 3 N. mucosa strains, 3 N. cinerea strains, 2 N. gonorrhoea strains, 2 N. sicca strains, 2 N. subflava strains, 1 N. lactamica strain, and 1 N. polysaccharea strain. Among them, 9 showed positive results in gonococcal fluorescence-labeled multiplex-PCR detection, 1 in cppB gene reaction, 5 in orfl gene reaction, and 3 in nspA gene reaction. The consistency rate was 85.7% between the above results from our gene detection and those from the routine bacteriological methods.</p><p><b>CONCLUSION</b>The cppB gene is absent in the non-gonococcal strains of Neisseria spp. that can cause male genitourinary tract infection. Most of the strains not only lack virulence-associated orfl and nspA genes, but also show positive results in gonococcal fluorescence-labeled multiplex-PCR detection, which is one of the important reasons for the misdiagnosis and missed diagnosis of gonorrhea infection. The combination of routine bacteriological methods and gene detection in laboratory examinations may help improve the accuracy rates of Neisseria species identification and clinical diagnosis of the infections caused by Neisseria spp.</p>

Study on the integration site and arrangement of SfII and SfX prophages in Shigella flexneri serotype 2b strains / 中华流行病学杂志

Objective To study the integration site and arrangement of SfII and SfX prophages in Shigella flexneri serotype 2b strains. Methods A series of primers were designed based on potential integration site of SfII and SfX prophages in Shigella flexneri serotype 2b strains, and PCR were performed for 50 serotype 2b strains to amplify special genes located in host and prophages. PCR products were sequenced to identify integration sites and arrangement of SfII and SfX. Results In all the serotype 2b strains, prophage SfII and SfX were adjacent to each other, and integrated into the thrW tRNA gene of the host, which were located between genes proA and yaiC of host. Prophage SfX was located immediately upstream of prophage SfII in all the detected 50 serotype 2b strains exception for strain 51251. Conclusion This was the first report on the integration site and arrangement of serotype-converting prophages SfII and SfX in Shigella flexneri 2b strains.

Secretion expression of cholera toxin B subunit in food-grading Lactococcus lactis expression system / 中华流行病学杂志

<p><b>OBJECTIVE</b>To clone and secretion express cholera toxin B subunit (CTB) in food-grading Lactococcus lactis expression systems.</p><p><b>METHODS</b>ctB fragment that encoding CTB was amplified by polymerase chain reaction (PCR) using the genomic DNA of Vibrio cholera strain 569B as template and was inserted into two secretion expression vector pSQZ and pSQ to construct food-grading expression system L.lactis MBP71/pSQZ-ctB and L.lactis MBP71/pSQ-ctB. The expressed CTB was detected by Western-blot assay.</p><p><b>RESULTS</b>The ctB fragment was successfully amplified from Vibrio cholera strain 569B and inserted into two secretion expression vectors pSQZ and pSQ to construct food-grading expression system L. lactis MBP71/pSQZ-ctB and L. lactis MBP71/pSQ-ctB. Western-blot assay demonstrated that CTB was secretion and expressed from L.lactis MBP71 harboring vectors pSQZ-ctB and pSQ-ctB, and the quantity of CTB secreted by L. lactis MBP71/pSQ-ctB was about 2 microg/ml, higher than that of L. lactis MBP71/pSQZ-ctB.</p><p><b>CONCLUSION</b>CTB was successfully secreted and expressed by food-grading L. lactis expression systems.</p>

Development of a protocol on pulsed field gel electrophoresis analysis for Streptococcus suis / 中华流行病学杂志

Objective To develop a PFGE protocol for Streptococcus suis.Methods We developed and optimized a PFGE protocol for S.suis,in terms of plug preparation,choice of restriction endonucleases and optimized electrophoresis parameters.By analyzing the genome sequences of S.suis P1/7 with Mapdraw of DNAStar.we found three restriction enzymes,Swa Ⅰ,Sma Ⅰ and Apa Ⅰ,were more suitable than others.Results Analysis of 100 isolates of S.suis including 34 of 35 serotypes identified,59,53 and 43 patterns were obtained from Swa Ⅰ,Sma Ⅰ and Apa Ⅰ restriction,respectively.The enzyme Swa Ⅰ had the greatest power for discrimination ability.Conclusion By optimization of the protocol at various conditions,a rapid,reproducible,economic and practical PFGE method for S.suis was developed.

Molecular epidemiology of enterohaemorrhagic Esacherichia coli O157 in some areas in China / 中华流行病学杂志

<p><b>OBJECTIVE</b>To understand the epidemiological characteristics of enterohaemorrhagic Escherichia coli (EHEC) O157 and to determine the degree of its genetic relations.</p><p><b>METHODS</b>Polymerase chain reaction (PCR) techniques and chromosomal DNA digested by restriction enzyme Xba I according to PulseNet directions by pulsed field gel electrophoresis (PFGE) method were applied to 300 E. coli O157 strains isolated from patients and animal sources from 1988 to 2005 from Henan, Jiangsu and Anhui provinces.</p><p><b>RESULTS</b>Very high prevalence of stx2 gene in EHEC O157:H7 strains isolated from some provinces of China was found and variation existed in some strains. We got 161 PFGE patterns from 300 strains. The stx2-producing strains could be clearly separated from stx2 variation-producing strains.</p><p><b>CONCLUSION</b>The variability of restriction enzyme-digestion patterns of O157 genomes suggested that the presence of some genomic diversity among the strains did exist.</p>

Immunoproteomic assay of secretive proteins from Streptococcus suis type 2 strain SC84 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To identify antigenic proteins secreted by Streptococcus suis (S. suis) type 2 strain SC84.</p><p><b>METHODS</b>Two-dimensional electrophoresis (2-DE), western-blot assay and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis were performed to search and identify antigenic proteins secreted by S. suis strain SC84, which triggered an outbreak of the disease in Sichuan province,China, in 2005.</p><p><b>RESULTS</b>A total number of 14 western blot spots were found on PVDF membrane. 11 spots which could be found the existence of matching protein on coomassie G-250-stained 2-DE gel were identified by MALDI-TOF MS. The 11 proteins, all located at extra-cellular or cell wall, were classified into 8 kinds of proteins. Among of them, muramidase-released protein (MRP), suilysin (Sly) and extra-cellular factor (EF) were the known antigenic proteins, but several proteins such as putative 5'-nucleotidase, ribo-nucleases G and E, and predicted metal-loendo-peptidase were newly found antigenic proteins. All the identified protein were found to have had the coding gene in genomic of S. suis strain 05ZYH33, isolated from patients in Sichuan province, China in 2005.</p><p><b>CONCLUSION</b>The newly found proteins could be used as voluntary antigens for detection and vaccination of S. suis.</p>

Identification of harboring stx2::IS1203 Escherichia coli O157:H7 strains isolated in China / 中华流行病学杂志

<p><b>OBJECTIVE</b>To understand the variation of Shiga toxin (stx) genes of Escherichia coli O157:H7 strains isolated in China.</p><p><b>METHODS</b>Polymerase chain reaction (PCR) was used to identity the types of stx genes and the nucleotide sequences of the amplified stex variants genes were determined. Compare to the cytotoxicity of Stx,variants were tested by HeLa cell assay.</p><p><b>RESULTS</b>We found novel stx2 genes in 3 of 289 strains of Shiga toxin-producing E. coli O157:H7 isolated from 1999 to 2002 in China. The novel stx2 genes were inserted by a 1.3-kb insertion sequence (IS) and the nucleotide sequences of IS showed 100% homology with that of IS1203 variant (IS1203v). The IS1203v inserted in the stx2 genes of three E. coli O157:H7 strains at different sites and the direction of the open reading frames (ORFs) of IS1203v of each strain was different. In addition to the above mentioned findings, the nucleotide sequences of three stx2 genes were completely identical and the type of the three Stx2 was Stx2 prototype. Compare to the cytotoxicity of Stx2 prototype, the novel Stx2 was found to be obviously lower.</p><p><b>CONCLUSION</b>E. coli O157:H7 strains harboring stx2::IS1203v genes were isolated in China. Consequently, the results of HeLa cell assay showed that the insertion of IS1203v could lead to low cytotoxicity of Stx2.</p>

Study on molecular typing by pulsed-field gel electrophoresis of food Listeria monocytogenes isolates from eight province (municipalities) of China / 中华流行病学杂志

<p><b>OBJECTIVE</b>To analyse the molecular types of Listeria (L.) monocytogenes, and to construct the standard China L. monocytogenes pulsed-field gel electrophoresis (PFGE) subtyping database, using the international standardized L. monocytogenes-PFGE protocol.</p><p><b>METHODS</b>118 L. monocytogenes strains isolated from 8 provinces or municipalities of China were subtyped according to L. monocytogenes-PFGE protocol.</p><p><b>RESULTS</b>118 strains of L. monocytogenes were divided into 39 subtypes. In the 39 subtypes, 37 strains (31.36%) were GX6A160004 pattern, showing it was the predominant Lm subtype in China.</p><p><b>CONCLUSION</b>Data from molecular typing suggested that the predominant Lm strains were distributed in different regions of China. PulseNet China-Lm database was constructed which was valuable for the molecular subtyping-based surveillance of L. monocytogenes.</p>

An outbreak of human Streptococcus suis serotype 2 infections presenting with toxic shock syndrome in Sichuan, China / 中华流行病学杂志

<p><b>OBJECTIVE</b>In mid-July 2005, five patients presented with septic shock to a hospital in Ziyang city in Sichuan, China, to identify the etiology of the unknown reason disease, an epidemiological, clinical, and laboratory study were conducted.</p><p><b>METHODS</b>An enhanced surveillance program were established in Sichuan, the following activities were introduced: active case finding in Sichuan of (a) laboratory diagnosed Streptococcus suis infection and (b) clinically diagnosed probable cases with exposure history; supplemented by (c) monitoring reports on meningococcal meningitis. Streptococcus suis serotype 2 infection was confirmed by culture and biochemical reactions, followed by sequencing for specific genes for serotype and virulence factors.</p><p><b>RESULTS</b>From June 10 to August 21, 2005, 68 laboratory confirmed cases of human Streptococcus suis infections were reported. All were villagers who gave a history of direct exposure to deceased or sick pigs in their backyards where slaughtering was performed. Twenty six (38%) presented with toxic shock syndrome of which 15 (58%) died. Other presentations were septicaemia or meningitis. All isolates were tested positive for genes for tuf, species-specific 16S rRNA, cps2J, mrp, ef and sly. There were 136 clinically diagnosed probable cases with similar exposure history but incomplete laboratory investigations.</p><p><b>CONCLUSION</b>An outbreak of human Streptococcus suis serotype 2 infections occurred in villagers after direct exposure to deceased or sick pigs in Sichuan. Prohibition of slaughtering in backyards brought the outbreak to a halt. A virulent strain of the bacteria is speculated to be in circulation, and is responsible for the unusual presentation of toxic shock syndrome with high case fatality.</p>