New EMA real-time fluorescence PCR method for detection of alive Listeria monocytogenes in foods / 中国人兽共患病学报

A new EMA real-time fluorescence PCR method was developed to detect alive Listeria monocytogenes in foods.The specific primers and probe were designed based on the conserved inlA gene.The pretreatment conditions including EMA of different concentrations and irradiating times were optimized.The detection limit and inhibition rate to dead bacteria of this method were confirmed by using direct plating method.The detection specificity was evaluated by using 35 L.monocytogenes strains,25 non-L.monocytogenes strains and 92 non-Listeria strains.Simulation detection experiments were performed on 15 beverage samples and 15 cooked meat samples supplemented separately with inactivated L.monocytogenes,alive L.monocytogenes and Staphylococcus aureus.Results showed that the Ct of EMA real-time fluorescence PCR for alive L.monocytogenes was Ct=38.46-3.30 × log (R2=0.999).The detection limit was 55 cfu per reaction.Inhibition rate of DNA of inactivated strains was over 99.98％.The Ct of 35 L.monocytogenes strains were between 16.21 and 29.38,while 25 non-L.monocytogenes strains and 92 non-Listeria strains had Ct ＞35.The variation coefficient of CT was less than 5％ when the experiments were repeated.Results of 30 simulation samples were consistent with that by using standard method.The test time by using newly developed EMA real-time fluorescence PCR was shortened from 3-5 days to about 10 h.The newly developed EMA real-time fluorescence PCR method for alive L.monocytogenes is rapid,convenient,specific and sensitive and could be applyed in foods inspection.

Foodborne pathogens in retail oysters in south China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the occurrence of important foodborne pathogens in shellstock Pacific oysters in the food markets in South China.</p><p><b>METHODS</b>From July 2007 to June 2008, retail oysters were collected in different seasons from South China and analyzed for the prevalence and levels of Listeria monocytogenes, Vibrio vulnificus and Vibrio parahaemolyticus.</p><p><b>RESULTS</b>None of L. monocytogenes could be detected in any of the 202 oyster samples tested, while E vulnificus and V. parahaemolyticus could be detected in 67 (54.9%) and 109 (89.3%) of the 122 oyster samples analyzed, respectively, with an MPN (most probable number) value greater than or equal to 3. V. vulnificus and V. parahaemolyticus with a more than 102 MPN/g were found in 36 (29.5%) and 59 (48.4%) of the 122 oyster samples, respectively. The tdh and trh genes were detected in 4 (0.3%) and 8 (0.6%) of the 1 349 V parahaemolyticus isolates, respectively. Of the 122 samples, 4 (3.3%) was positive for either tdh or trh. The levels of V. vulnificus and total V. parahaemolyticus in oysters in South China varied in different seasons.</p><p><b>CONCLUSION</b>V. vulnificus and pathogenic V. parahaemolyticus are frequently found in oysters in south China, which may pose a potential threat to public health. Data presented here will be useful for the microbiological risk assessment in oysters in China.</p>

Study on the difference of genes and the type identification of hantavirus from Lishui,Zhejiang province / 中华流行病学杂志

Objective To isolate hantavirus from Lishui county-one of the epidemic regions for hemorrhagic fever with renal syndrome(HFRS),in Zhejiang province,and to identify the serotype and molecular/biological characteristics of a new HTN subtype hantavirus(HV)strains,hopefully to provide evidence for HFRS prevention and therapy.Methods Data on the host animals was collected from Lishui,Zhejiang province in 2007.Direct immunofluorscece assay was adopted to determine HFRS antigens and the lung tissues from HV infeeted Vero-E6 cells for HV isolation,then total RNA was extracted from Hantavirus Lishui strains and amplified by RT-PCR M,S segments of strains genome were also clened and sequenced and compared with those of other strains of HV Results 2 strains virus(ZLS6-11 and ZLS-12)Were successfully isolated from 7 positive lung samples of mice and were identified as HTNV by anti-McAb and phylogenetic analysis.With sequence compation.We found that 2 strains with complete M and S segment had higher homology with HTN-type strains than with other types of HV,but 13.4%-20.7%and 10.3%-16.1%of the genes were found which were difierent from HTNV.The phylogenetic trees constructed by complete S and M segment showed that ZLS6-11 and ZLS-12 strains were located in HTNV group,and structured independent embranchment.Conclusion ZLS6-11 and ZLS-12 Strains were believed to belong to HTN-type and phylogenetically different from the HTNV.

Establishment and application of loop-mediated isothermal amplification method for rapid detection of Legionella pneumophila / 中华流行病学杂志

Objective To develop a loop-mediated isothermal amplification (LAMP) method for rapid diagnosing of Legionella pneumophila in the Pathogen Detection Department(PDD) or in small-scale laboratory. Methods Five primers (2 Inner Primers, 2 Outer Primers and a Loop Primer) for the LAMP test were designed by targeting the mip gene of Lpneumophila and reaction system of LAMP reaction was optimized. 12 strains of L.pneumophila, 45 local strains, 6 non-L.pneumophila strains, 11 other strains and 59 environmental water samples were analyzed to evaluate the specificity and sensibility of the LAMP amplification. At the same time, the results of the LAMP were also compared with biochemical culture and quantitative PCR methods. Results The amplification products of L.pneumophila turned green by visual inspection and had ladder-like pattern on the gel, but non-L.pneumophila and other products from the strains remained orange by visual examination and had no band on the gel. The detection rate of LAMP was higher than the biochemical culture and the real-time PCR methods. Reaction time of the LAMP method was only 1.5 h and the detection limit of LAMP assay was 5 cfu/reaction. In addition, the LAMP results could be determined only by visual inspection. Conclusion LAMP assay targeting the mip gene of L.pneumophila appeared to be rapid, specific, and sensitive for the detection of L.pneumophila. This method not only reduced the dependence of complicated equipment but also had a potential for wider use in the PDD, small-scale laboratory, emergency motor vehicle or for field survey.

Molecular characterization and sequence comparison of the M and S segments of Hantavirus ZJ5 strain / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>In order to understand the molecular characters of Hantavirus ZJ5 strain, its complete M and S genome were sequenced and compared with that of other hantavirus strains.</p><p><b>METHODS</b>We prepared the total RNA from ZJ5. Infected cells and the raw or purified RT-PCR product was cloned and sequenced.</p><p><b>RESULTS</b>With sequence compation, we found ZJ5 strain complete M and S segment had higher homology with SEO-type strains than other type of HV, but differential genes were 11.7%-19.2% and 6.7%-14.5% from SEOV. The phylogenetic trees constructed by complete M ind S segment showed that ZJ5 strain was located in SEOV group, and structured alone embranchment.</p><p><b>CONCLUSION</b>The ZJ5 strain was believed to belong to SEO-type virus,and suggest that ZJ5 strain is a new subtype S SEOV group,and structured alone embranchment.</p><p><b>CONCLUSION</b>The ZJ5 strain was believed to belong to SEO-type virus, and suggest that ZJ5 strain is a new subtype from other SEO viruses.</p>

Molecular biological analysis of genotyping and phylogeny of severe acute respiratory syndrome associated coronavirus / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>SARS-CoV is the causative agent of severe acute respiratory syndrome (SARS) which has been associated with outbreaks of SARS in Guangdong, Hong Kong and Beijing of China, and other regions worldwide. SARS-CoV from human has shown some variations but its origin is still unknown. The genotyping and phylogeny of SARS-CoV were analyzed and reported in this paper.</p><p><b>METHODS</b>Full or partial genomes of 44 SARS-CoV strains were collected from GenBank. The genotype, single nucleotide polymorphism and phylogeny of these SARS-CoV strains were analyzed by molecular biological, bioinformatic and epidemiological methods.</p><p><b>RESULTS</b>There were 188 point mutations in the 33 virus full genomes with the counts of mutation mounting to 297. Further analysis was carried out among 36 of 188 loci with more than two times of mutation. All the 36 mutation loci occurred in coding sequences and 22 loci were non-synonymous. The gene mutation rates of replicase 1AB, S2 domain of spike glycoprotein and nucleocapsid protein were lower (0.079% - 0.103%). There were 4 mutation loci in S1 domain of spike glycoprotein. The gene mutation rate of ORF10 was the highest (3.333%) with 4 mutation loci in this small domain (120 bp) and 3 of 4 loci related to deletion mutation. By bioinformatics processing and analysis, the nucleotides at 7 loci of genome (T:T:A:G:T:C:T/C:G:G:A:C:T:C) can classify SARS-CoV into two types. Therefore a novel definition is put forward that according to these 7 loci of mutation, 40 strains of SARS-CoV in GenBank can be grouped into two genotypes, T:T:A:G:T:C:T and C:G:G:A:C:T:C, and named as SARS-CoV Yexin genotype and Xiaohong genotype. The two genotypes can be further divided into some sub-genotypes. These genotypes can also be approved by phylogenetic tree of three levels of 44 loci of mutation, spike glycoprotein gene and complete genome sequence. Compared to various strains among SARS-CoV Yexin genotype and Xiaohong genotype, GD01 strain of Yexin genotype is more closely related to SARS-CoV like-virus from animals.</p><p><b>CONCLUSION</b>The results mentioned above suggest that SARS-CoV is responding to host immunological pressures and experiencing variation which provide clues, information and evidence of molecular biology for the clinical pathology, vaccine developing and epidemic investigation.</p>