Search

To balance the nutrition and improve quality of sausages. This research investigated quality improvements of Sichuan sausages by 41.5% poultry, rabbit meat and fish addition. The moisture, crude fat, crude protein contents, pH, Aw, colour, texture, TBARS, protein carbonyl, free thiol groups, fatty acids composition and sensory attributes of pork, poultry, rabbit meat and fish mixture group sausages were evaluated. The results showed that the hardness of pork and rabbit meat mixture group sausages (76.37, 68.01 kg) was significant (P

Objective: To establish a method for the determination of shrimp allergens in meat products by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Methods: Meat products (cooked ham, sausages and meatballs) with complex matrix components were selected as the detection target. The samples were extracted by ultrasonic in phosphate buffer solution (PBS) for 30 min, and acetonitrile was added to remove fat after centrifugation (10000 r/min, 15 ℃, 10 min). Finally, trypsin (1 mg/mL, 10 μL) was added to the sample diluents which had been mixed with internal peptide (2.5 μmol/L, 40 μL), and the enzymolysis process lasted 16 h at 37 ℃ before UPLC-MS/MS analysis. The samples were separated on a T3 column, and eluted by gradient elution with 0.1% formic acid aqueous solution and acetonitrile. The data was collected by multiple reaction monitoring (MRM) in positive ion mode, and quantified by internal standard method. Results: The method was used to determine the content of shrimp allergen in meat products, and the quantitative peptide had a good linear relationship with the determination coefficients 1.0000 in the range of 0.001~2.0 μmol/L. The limits of detection was 0.67 mg/kg, and the limits of quantification was 2.00 mg/kg. The recovery was 83.2%~104.5% and the precision was 2.63%~7.92% at three spiked levels. Conclusion: The method has the advantages of strong specificity, high sensitivity and good recovery. It was suitable for the screening and quantification of shrimp allergens in meat products.

With the characteristics of large space-time and satellite-ground network integration, the space integrated ground network has attracted much attention.Satellites can not only be used as emergency communication supplements, but also serve as air stations to expand the coverage of terrestrial networks, occupying an important position in both military and civilian scenarios.The entity authentication and key negotiation mechanism can prevent the malicious entities from joining the integrated network to steal users’ privacy, and guarantee network information security.In view of the characteristics of the high satellite-ground transmission delays, exposed links, limited processing capability and dynamic topology of the integrated network, a lightweight authentication scheme between satellites and ground suitable for double-layer satellite network was proposed to achieve a secure satellite networking architecture with session keys to protect data transmission.The scheme was based on symmetric cryptographic system, using lightweight cryptographic algorithms and introducing group key and hierarchical management mechanisms.The proposed scheme included three parts: inter-satellite authentication for geostationary earth orbit satellites, layer and inter-satellite authentication for same low earth orbit, and inter-satellite authentication for adjacent low earth orbit satellites.The group key and hierarchical management mechanism improved the efficiency of inter-group information transfer, reduced the authentication pressure on the ground control center, and enhanced the authentication security strength by realizing double verification in the three-entities authentication protocol.Different from the previous single scene authentication, the proposed authentication protocol took the form of multiplexing authentication parameters, which can realize the authentication requirements of dual scenes in one process.The results of Scyther, a protocol formal security simulation tool, show that the proposed scheme achieves secure access authentication.Compared with existing protocols, the proposed scheme improves authentication security and reduces communication and computational overhead.

Post-drilling evaluation is an important part of prospect evaluation in the oil and gas exploration processes. With the comparative analysis of pre-drilling prediction and post-drilling results, the experience is summarized, and the deficiencies in pre-drilling evaluation and prediction are corrected and improved, so that the pre-drilling evaluation and prediction results of exploration targets are more accurate, and the success rate for the coming oil and gas exploration can be improved. Based on introducing the main contents and methods of post-drilling evaluation, the problems and solutions that need to be focused on in post-drilling evaluation are summarized in this paper. By analyzing the geological risk factors and reserve parameters of both successful and failed drilling, the key elements of large deviation between pre-drilling prediction and post-drilling estimation are compared, and the causes for the deviation are discussed and improved, which can be helpful on improving the geological explorers' ability of prediction and enhancing the success ratio of exploration in the following work.

Low activation ferritic martensitic steel is considered as one of the candidate structural materials for the fourth generation reactor and fusion reactor. Low activation ferritic martensitic steel is based on Fe-Cr alloy, in which W is an important solute element. Irradiation embrittlement at low temperature is one of the important problems limiting the service of low-activation ferrite martensitic steels. The precipitated clusters during the service process cause hardening and embrittlement of the material due to the obstruction of dislocation movement by the precipitated clusters. An in-depth understanding of cluster precipitation behavior during service is helpful to understand the irradiation embrittlement of low-activation ferrite martensitic steels at low temperature. In order to simulate the precipitation kinetics of Fe-Cr alloy, by introducing composition-dependent pair potential, some improvements were made on the MIET_AKMC software developed by ourselves. The precipitation kinetics of coherent Cr-rich precipitates/clusters in Fe-Cr(8%, 10%, 16%, 20%)-W(0%, 1%, 2%) alloys during thermal ageing was simulated using the atomic kinetic Monte Carlo (AKMC) method. After thermal ageing, Cr precipitate into clusters to form Cr-rich precipitates/clusters, while W is still in a solid solution state. At the beginning of coarsening process, the radius of clusters, which was about (0.57±0.03) nm, was independent on simulation temperature and the initial Cr content in the box. The precipitation process of Cr cluster can be divided into three stages: nucleation, growth and coarsening. The Cr concentration in box and simulation temperature have an influence on the precipitation rate of Cr clusters. The presence of W can delay the precipitation kinetic process and the effect is most significant when Cr content is about 10%. This is due to that when Cr content is about 10%, the absolute value of the short-range order parameter for Fe-Cr has a maximum, quite independent of the composition and the temperature. The delay effect of W was related with the stronger binding energy between W and vacancy. For the binding energy between Cr and vacancy is 0.06 eV, while the binding energy between W and vacancy is 0.14 eV. From the simulation results, it can be deduced that W is benefit for materials performance, expect for solution strengthening, it can delay the precipitation process of Cr-rich clusters, which is harmful for materials performance.

In order to solve the current problems of electronic invoices, such as anti-counterfeiting, privacy leakage and low verification efficiency, an electronic invoice public verification architecture based on the national signature algorithm was proposed.Electronic invoice documents have complex data sources, sensitive user information on the ticket surface, frequent data flow and other characteristics as well as the demand for efficient public verification.Based on these motivations, the e-invoice generation and verification protocol was designed.Besides, a signature code generation scheme for invoice anti-counterfeiting was proposed based on certificate-free joint signature.Then multiple data verification and signatures of the invoicing party and the taxation authority can be realized.All kinds of invoice holders can not only verify the authenticity and data integrity of electronic invoices, but also publicly check the authenticity and data integrity of electronic invoices.The integration of data encryption algorithms protects user privacy data in electronic invoices, and at the same time can fulfill the requirements for authenticity or status verification under various invoice application scenarios.The checking party only needs to verify one signature to confirm the authenticity of the electronic invoice signed by both parties.Scyther security simulation tools were used to analyze the security of the proposed solution, which can verify the integrity and authenticity of the data source and check the confidentiality of the privacy data under various types of attacks.Compared with a typical overseas e-invoice checking scheme and a similar digital signature-based e-invoice checking scheme, the proposed scheme has obvious advantages in terms of checking efficiency and invoice file size.

Social network has gradually become the main way in communication with the development of informa-tion technology and the issue of privacy preserving in social network has also obtained more and more attention due to the interactive information exposed in the open social networks.Firstly,an overview of the concept of social net-work and privacy was givem.Secondly,the current mainly two methods:anonymity mechanism and access control technique employed for privacy preservation in social networks were reviewed and discussed.Finally,the vulner-abilities and challenges of the current mechanism were analyzed and the potential research issues for the future re-search works were showed.

This study established an HPLC fingerprint and multi-component content determination method for salt-fired Eucommiae Cortex, and evaluated the quality of salt-fired Eucommiae Cortex from different sources using fingerprint similarity evaluation, cluster analysis(CA), principal component analysis(PCA), and orthogonal partial least square discriminate analysis(OPLS-DA). HPLC was launched on a Cosmosil 5C_(18)-MS-Ⅱ column(4.6 mm×250 mm, 5 μm) by gradient elution with a mobile phase of methanol-0.2% phosphoric acid aqueous solution at a flow rate of 1.0 mL·min~(-1), detection wavelength of 238 nm, column temperature of 30 ℃, and an injection volume of 10 μL. The results of fingerprint similarity evaluation for 20 batches of salt-fired Eucommiae Cortex indicated that, except for batch S3 with a similarity of 0.893, the similarity of the other 19 batches was of ≥ 0.919, suggesting good similarity. Fourteen common peaks were calibrated and seven common peaks were identified including geniposidic acid. The mass fractions of geniposidic acid, chlorogenic acid, geniposide, genipin, pinoresinol diglucoside, liriodendrin, and pinoresinol-4-O-β-D-glucopyranoside were 0.062 0%-0.426 9%, 0.024 9%-0.116 5%, 0.009 5%-0.052 9%, 0.005 5%-0.034 8%, 0.115 9%-0.317 8%, 0.016 4%-0.108 8%, and 0.026 4%-0.039 8%, respectively. Using CA, PCA, and OPLS-DA, the 20 batches of salt-fired Eucommiae Cortex were classified into three categories. Additionally, through the analysis of variable importance in projection(VIP) under OPLS-DA, two differential quality markers, geniposidic acid and chlorogenic acid, were identified. The established HPLC fingerprint and multi-component content determination method is stable and reliable, providing a reference for quality control of salt-fired Eucommiae Cortex.

Objective: To review the effectiveness and safety of auriculotherapy in treatment of insomnia., Methods: The articles were collated by computer retrieval from 8 databases from the initiation to April 30, 2021, i.e. PubMed, EMbase, the Cochrane Library, the Web of Science, CNKI, Wanfang database, VIP and Chinese biomedical literature database (SinoMed). Meta-analysis was conducted with RevMan5.3 software., Results: A total of 38 articles were included, with 3 707 cases involved. The results showed: ①The effective rate of auriculotherapy was better than that of single application of western medication with sleeping pills ( RR =1.26, 95% CI :1.15 to 1.39, Z =4.77, P <0.000 01), conventional acupuncture ( RR =1.10, 95% CI : 1.05 to 1.16, Z =3.83, P =0.000 1) and Chinese herbal medicine ( RR =1.41, 95% CI: 1.23 to 1.63, Z= 4.80, P <0.000 01), respectively. ② The total score of PSQI was reduced remarkably under the auriculotherapy when compared with the single application of western medication ( MD= -1.61, 95% CI : -2.61 to -0.60, Z =3.14, P= 0.002) and Chinese herbal medicine ( MD= -3.76, 95% CI : -4.84 to -2.68, Z =6.84, P <0.000 01). But the difference was not significant when compared with conventional acupuncture ( MD =-1.02, 95% CI : -2.11 to 0.08, Z =1.82, P =0.07). ③Auricular point selection: the auricular points distributed in the areas of vagus nerve were more advantageous in reducing PSQI score ( MD =-3.21, 95% CI : -4.45 to -1.96, Z =5.03, P <0.000 01) compared with the points in other areas. ④Stimulant selection: the difference in the effective rate was not significant among different stimulants (magnetic beads, the seeds of Vaccaria segetalis , micro-needles) ( MD =1.62, 95% CI : 0.71 to 3.73, Z =1.14, P =0.25). ⑤Stimulation frequency: there was no significant difference between high-frequency stimulation and low-frequency stimulation of auricular point sticking in improving the effective rate and reducing PSQI score ( P >0.05). But the result should be considered cautiously in terms of the sensitivity analysis. ⑥Adverse reactions: the case numbers of adverse reactions of auriculotherapy (auricular point sticking) were less than those of western medication ( MD =0.15, 95% CI : 0.06 to 0.35, Z =4.38, P <0.000 1)., Conclusion: Auriculotherapy has certain curative advantages in treatment of insomnia compared with western and Chinese medications as well as conventional acupuncture. This therapy may relieve the symptoms of insomnia and has less adverse effects. But those outcomes need to be further verified with more high-quality randomized controlled trials.

Objectives: To investigate the psychological and behavioral problems and related influencing factors in children and adolescents during the coronavirus disease 2019 (COVID-19) epidemic., Methods: China National Knowledge Infrastructure, Wanfang Data, PubMed, and Web of Science were searched using the method of subject search for articles published up to March 31, 2022, and related data were extracted for Scoping review., Results: A total of 3 951 articles were retrieved, and 35 articles from 12 countries were finally included. Most of the articles were from the journals related to pediatrics, psychiatry, psychology, and epidemiology, and cross-sectional survey was the most commonly used research method. Psychological and behavioral problems in children and adolescents mainly included depression/anxiety/stress, sleep disorder, internet behavior problems, traumatic stress disorder, and self-injury/suicide. Influencing factors were analyzed from the three aspects of socio-demographic characteristics, changes in living habits, and ways of coping with COVID-19., Conclusions: During the COVID-19 epidemic, the psychological and behavioral problems of children and adolescents in China and overseas are severe. In the future, further investigation and research can be carried out based on relevant influencing factors to improve the psychological and behavioral problems.

Objective: To observe the effect of five-element acupuncture on the cognitive function repair of migraine patients with depression/anxiety disorder., Methods: The migraine patients with depression/anxiety disorder (19 cases, 5 cases dropped off) were taken as the observation group, and received five-element acupuncture twice a week for 8 weeks. Healthy subjects (19 cases) were selected by demographic data matching as the control group. The cognitive function was evaluated with the event related potential (ERP) technique, and the latency and amplitude of visual evoked potential P300 were adopted as the observation indexes. The headache days (every 4 weeks), headache intensity [visual analogue scale(VAS) score], and headache impact test-6 (HIT-6) score, Hamilton depression scale (HAMD) score and Hamilton anxiety scale (HAMA) score were used as the observation indexes for curative effect., Results: Before the treatment, latency of target stimulus at Fz [ (417.5±34.3) ms] in the observation group was extended compared with the healthy subjects of the control group [(388.6±42.1) ms, P <0.05]. In the observation group, the latency of each point target stimulus [Fz: (376.1±36.2) ms, F3: (374.8±37.6) ms, F4: (372.0±37.6) ms] after treatment were shorter than those [Fz: (417.5±34.3) ms, F3: (417.4±33.8) ms, F4: (416.0±36.6) ms] before treatment ( P <0.05). Before and after treatment, there was no significant difference in the amplitude of each point between the observation group and the control group ( P >0.05). In the observation group, the headache days was shorter than that before treatment ( P <0.01), and the VAS score, HIT-6 score, HAMD score and HAMA score were all lower than before treatment ( P <0.01)., Conclusion: There are some cognitive impairments in migraine patients with depression/anxiety disorder. Five-element acupuncture not only relieves headache, anxiety and depression effectively, but also improves the activation level of the frontal lobe. It significantly repairs the impaired cognitive function.

Objective: To observe the impacts on skeletal fluorosis pain, joint motor dysfunction and urine fluoride excretion in the treatment with fire needle therapy, electroacupuncture and calcium carbonate D3., Methods: The randomized controlled trial was adopted. Ninety-five patients were randomized into a fire needle group (31 cases), an electroacupuncture group (33 cases) and a calcium carbonate D3 group (31 cases). In the fire needle group and the electroacupuncture group, Ashi points, Dazhui (GV 14), Geshu (BL 17), Quchi (LI 11), Hegu (LI 4), Xuehai (SP 10) points were selected and stimulated with fire needle and electroacupuncture separately, three times a week. In the calcium carbonate D3 group, calcium carbonate D3 tablets was prescribed for oral administration, 600 mg each time, twice a day. The duration of treatment was 2 months in the electroacupuncture group and calcium carbonate D3 group and 1 month in the fire needle group. VAS score, the range of motion (ROM) and urine fluoride value were compared before and after treatment in the patients of the three groups., Results: After treatment, VAS value and ROM were improved significantly in the patients of the three groups (all P < 0.05), the difference was not significant in comparison of the three groups (all P > 0.05). After treatment, the urine fluoride value was increased significantly in the fire needle group [(7.89 +/- 3.61) mg/L vs (9.81 +/- 4.17) mg/L, P < 0.01] and was increased in the electroacupuncture group [(7.53 +/- 3.46) mg/L vs (8.97 +/- 4.21) mg/L, P < 0.05]. The difference was not significant in comparison before and after treatment in the calcium carbonate D3 group (P > 0.05)., Conclusion: The fire needle therapy, electroacupuncture and calcium carbonate D3 all have the clinical value in the prevention and treatment of skeletal fluorosis and the difference in the therapeutic effect has not been discovered among them yet at present. But it has been found that the fire needle therapy and electroacupuncture display the active significance in the promotion of urine fluoride excretion.

Objective: To study the infection status and pathogenic features of human metapneumovirus (hMPV) among children with acute respiratory tract infection in Hangzhou., Methods: A total of 372 children less than 14 years old with acute respiratory tract infections were recruited as subjects from the pediatric clinic or intensive care unit (ICU) of 3 hospitals in Hangzhou during November 2009 to January 2010, and November 2010 to January 2011. A total of 372 specimens were collected, including 351 respiratory swab, 9 nasopharyngeal aspirate material, 8 endotracheal aspirate material and 4 sputum. The total nucleic acid was then extracted from the specimens, and the nucleoprotein (N) gene of hMPV was amplified by RT-PCR, whose positive products were sequenced and analyzed. Africa green monkey kidney cells (Vero-E6) were applied to culture hMPV among the positive samples; meanwhile fluorescence quantitative RT-PCR was adopted to test other respiratory virus infection., Results: Out of 372 patients, 42 (11.2%) were positive for N gene of hMPV. The positive rate of hMPV among boys was 11.5% (26/226), and correspondingly 10.9% (16/146) among girls. The difference showed no statistical significance (χ(2) = 0.026, P > 0.05). The youngest patient was only 2 month-old and the eldest patient was 14 years old. The median of the patients' age was 24 months. Fifteen positive samples amplified by RT-PCR were sequenced, and all turned out to be subtype B1; whose similarity to GD165 found in Guangdong was 98.1% - 99.5% and similarity to BJ1897 in Beijing was 87.8% - 89.2%. The co-infection rate between hMPV and other respiratory virus was 45.2% (19/42); most of which was between hMPV and respiratory syncytial virus, whose rate at 26.1% (11/42)., Conclusion: hMPV was the single genotype relevant with the acute respiratory tract infection disease among children in Hangzhou district; however, the co-infection with other respiratory virus did exist.

Objective: To observe the impacts of acupuncture of reinforcing kidney and activating spleen on the excretion of urinary fluoride and pain of the patients with drinking-water type fluorosis., Methods: The randomized controlled and single-blind trial was adopted. Seventy-two cases were randomized into an observation group and a control group, 36 cases in each one. In the observation group, acupuncture was applied at Pishu (BL 20), Shenshu (BL 23), Guanyuan (CV 4), Zusanli (ST 36), etc. , three treatments a week. In the control group, the Calcium Carbonate D3 tablets were prescribed for oral administration, 600 mg each time, twice a day. The duration of treatment was 2 months. The changes of the content of urinary fluoride and pain score (by VAS) before and after treatment between two groups were compared., Results: The urinary fluoride excretion was increased obviously after treatment in the observation group (P < 0.01), which was superior apparently to that in the control group [(11.06 +/- 4.54) mg/L vs. (8.30 +/- 4.14) mg/L, P < 0.05]. After treatment, VAS score was reduced significantly in either group (both P < 0.01). The result in the observation group was lower remarkably than that in the control group (1.93 +/- 1.30 vs. 3.47 +/- 2.29, P < 0.01)., Conclusion: Acupuncture achieves the significant efficacy on the promotion of urinary fluoride excretion and pain relieving of the patients with drinking-water type fluorosis in light of reinforcing kidney and activating spleen, which is superior to the oral administration of the calcium carbonate D3 tablets.

Objective: To investigate the heterozygous genotype and molecular characteristics of Organophosphorus resistance associated with heterozygous Estbeta2 of esterase B2 gene from natural population of Culex pipiens complex., Methods: Genomic DNA was extracted from natural populations of Culex pipiens complex in Hangzhou. The PCR-restriction fragment length polymorphism (PCR-RFLP) assay was applied to type the resistance associated esterase gene. Estbeta2 of esterase B2 gene was identified by PCR-RFLP, and the genotyping for heterozygous Estbeta2 was carried out after restriction enzyme digesting by Bfm I endonuclease., Results: The DNA was isolated from 207 Culex pipiens respectively, while 156 PCR samples showed positive and the positive rate was 75.36% (156/207). The PCR-RFLP assay of esterase B2 gene revealed that the Estbeta2 was accounted about 28.20% (44/156) in 156 positive samples. There were two genotypes identified, namely homozygous Estbeta2 (90.90%, 30/33) and heterozygous Estbeta2 (9%, 3/33), heterozygous Estbeta2 was in existence of a hybrid form as which combined with Estbeta2 and a subtype (Estbeta2/Estbeta2(1))., Conclusion: Heterozygous Estbeta2 of Organophosphorus resistance associated with esterase genotype was determined in natural population of Culex pipiens, and a genotyping method was established.

Objective: To develop a rapid and simple multiplex polymerase chain reaction (PCR) method which discriminates extended-spectrum beta-lactamases (ESBLs) genes in sporadic Shigella isolates from 1998 to 2007 in Hangzhou city, China., Methods: After ESBLs screening according to the Clinical and Laboratory Standards Institute (CLSI) method, CTX-M, TEM, SHV and OXA-1 encoding genes were detected by using a multiplex PCR method, and the results were verified by 8 single gene PCR amplification., Results: Seventeen isolates harbored ESBLs genes among 195 Shigella isolates (8.72%). Genes encoding CTX-M (17 strains), TEM (2 strains), OXA-1 (10 strains) and SHV (0 strains) were discriminated with multiplex PCR analysis, which coincided with eight single gene PCR analysis at 94.12%., Conclusion: Multiplex PCR should be a suitable tool for initial rapid screening and discriminating ESBLs genes in Shigella isolates. With similar trend of national surveillance data, the proportion of sporadic Shigella isolates harbouring ESBLs genes might probably be on increase.

Objective: To investigate the relationship between the mRNA expression levels of p53-mediating DNA damage and repair genes in the peripheral blood lymphocytes of workers and their exposures to benzene in their working environment., Methods: The mRNA expression levels of p53 and related genes were determined by SYBR Green I chimeric fluorescence quantitative real-time RT-PCR analysis in peripheral blood lymphocytes of 72 workers, who were classified into group A (46 direct exposure to benzene) and group B (26 indirect exposure to benzene) based on their positions, and 29 controls. The differences of gene expression levels were analyzed by software REST 2005. Meanwhile, the peripheral blood leukocytes, hemoglobin and platelet of workers and controls were counted. Benzene content was measured in the samples of toluene, used as raw material, and spraying agents and benzene, toluene and xylene concentrations in the air of workplaces were monitored., Results: There were no significant differences in the mRNA expression levels of p53, Ku80, Ape1 and Mdm-2 between group A or group B and control group (P > 0.05). The expression up-regulation of p21 mRNA was found, but without significant difference (P > 0.05). However, the mRNA expression levels of Rad51, Bcl-2, Bax, Xpa and Xpc in group A and Rad51 in group B were downregulated significantly (P < 0.05 or P < 0.01). Moreover, both the counts of white blood cell, hemoglobin and platelet in group A were (4.93 +/- 1.27) x 10(9)/L, (123.97 +/- 11.80) g/L and (124.02 +/- 41.22) x 10(9)/L respectively and platelet in group B (135.80 +/- 39.44) x 10(9)/L were significantly lower than in control group (P < 0.05 or P < 0.01)., Conclusion: The mRNA expression levels of some p53-mediating DNA damage and repair genes are downregulated in the workers chronically exposed to low benzene concentration. The working environment impacts on health of group A workers are greater than the ones of group B.

Objective: To develop a multiplex real-time PCR for the detection of Salmonella invasion protein A gene (invA), enterotoxigenic Escherichia coli (ETEC) heat-labile I enterotoxin gene (elt), and Shigella or enteroinvasive E. coli (EIEC) invasive plasmid antigen H gene (ipaH)., Methods: Under the optimized reaction conditions of the multiplex real-time PCR, invA, elt, and ipaH were determined in 10-fold series of dilution of DNA extracted from Salmonella enterica serovar Typhimurium, ETEC 44815 strain and Shigella F301 strain. The three genes were examined in 90 fecal samples from diarrhea patients using the multiplex real-time PCR. When PCR-positive samples were found, the target strains were isolated and identified., Results: The detectable concentration for this multiplex real-time PCR was 10 CFU/microl for Shigella F301 strain, 10(2) CFU/microl for S. enterica serovar Typhimurium and ETEC 44815 strain, respectively. Out of 90 fecal samples from diarrhea patients, thirteen were found positive for elt gene (14.4%), and five were found positive for ipaH gene (5.6%). Three E. coli strains positive for elt gene and four E. coli strains positive for ipaH gene were isolated successfully from the PCR-positive samples mentioned above. The detection of invA, elt and ipaH genes was completed in 10 h, which included an enrichment period of 6 h., Conclusion: The multiplex real-time PCR assay can detect invA, elt, ipaH simultaneously in a single reaction, moreover, it can detect for virulence genes in strains of Salmonella, ETEC, and Shigella or EIEC and screen these pathogens in fecal specimens from patients with diarrhea with a high specificity.

Objective: To investigate the genotypes , allele frequencies and dynamic distribution on resistance associated esterase genes of Culex pipiens complex in Hangzhou., Methods: The PCR-restriction fragment length polymorphism (PCR-RFLP) assay was applied to type the resistance associated esterase genes, and dynamic surveillance on frequencies of the resistance associated esterase gene of natural population of Culex pipiens complex in Hangzhou during 2003-2005, and phenotype of the resistance associated esterase genes were detected by esterase starch gel electrophoresis technique., Results: The PCR-RFLP assay of esterase allele genes for three consecutive years disclosed four esterase genotypes, namely, the world-wide highly active homozygous Est beta 1(1) (50%-54%), homozygous Est beta 2 (29%-34%), heterozygous Est beta 1(1)/beta 2 (5%-10%) and Est beta N (3.13%) of a new homozygous genotype. The research of the resistance associated esterase genes phenotype in natural population of Culex pipiens complex in Hangzhou in 2005 with esterase starch gel electrophoresis technique revealed four major types, namely, Est beta 1(1) (61%), Est alpha 2/beta 2 (12%), Est alpha 8/beta 8 (7%) and sensitive phenotype (29%)., Conclusion: There should be various resistance associated esterase genotypes in natural population of Culex pipiens complex in Hangzhou. During the period of 2003-2005, Est beta 1(1) was the major type; Est alpha 2/beta 2 was the second. Est beta N was a new esterase genotype detected in 2005 only with a mere percentage of 3.13%. As for its resistance to the new insecticide, a follow-up study should be needed. The molecular typing of the amplified esterase gene should be consistent with the resistance associated esterase genes phenotype.

Objective: To know the molecular characteristic of Shigella flexneri 4c isolates from patients in two food-poisoning outbreaks and one sporadic diarrhea case in Hangzhou, China., Methods: S. flexneri isolates from patients in two food-poisoning outbreaks (outbreak 1 and outbreak 2, n = 13 and n = 12, respectively) and one sporadic diarrhea patient (n = 1) in Hangzhou during 2003 and 2005 were serotyped. Antibiotic resistances of these isolates were measured by the Kirby-Bauer method. Invasive plasmid antigen gene ipaH was examined by PCR. Pulse field gel electrophoresis (PFGE) was performed for molecular typing., Results: In outbreak 1, all 13 isolates were S. flexneri 4c, of them 6 isolates tested were quite different in PFGE patterns with dice coefficient from 0.78 to 0.92. In outbreak 2, 10 isolates were S. flexneri 4c and 2 isolates were S. flexneri X, however their PFGE patterns were almost identical (dice coefficient > 0.8). Compared to the two outbreaks isolates, the sporadic isolate was demonstrated with a distinct PFGE pattern (dice coefficient < 0.8). The antibiotic resistance patterns with 14 kinds of antibiotics had a little difference among the isolates from outbreak 1, outbreak 2 and sporadic diarrhea patient, but the same pattern was found among 10 isolates of S. flexneri 4c and 2 isolates of S. flexneri X from outbreak 2., Conclusions: PFGE might distinguish the isolates from these two outbreaks and the sporadic diarrhea patient. Some differences in PFGE patterns, serotypes and antibiotic resistance patterns might occur among S. flexneri 4c isolates during an outbreak.

In order to validate the usefulness of gyrB genotype for the classification and identification of Vibrio cholerae and Vibrio parahaemolyticus isolates, the phylogenetic analysis of 13 V. cholerae, 8 V. parahaemolyticus, 2 Aeromonas hydrophila and 1 Plesiomonas shigelloides strains was carried out using the partial coding sequence of gyrB, a gene that encodes the B subunit of DNA gyrase (topoisomerase type II ) in bacteria. These strains were separately clustered at species level and typed by the DNA sequences of reference strains from GenBank. CtxA positive V. cholerae strains including 8 clincical isolates of 0139 and 2 clinical isolates of 01 formed one cluster. Four V. parahaemolyticus strains of 1 isolate from 2002 Zhejiang outbreak patient ( tdh positive), 2 clinical isoltates from 2004 and 1 strain from Japan were grouped with an environmental isolate ( trh positive) from 2001. GyrB genotype is applicable to species identification of V. cholerae, V. parahaemolyticus, A. hydrophila and P. shigelloides isolates. The ctxA positive 0139 and 01 group of V. cholerae are closely related, as reflected by gyrB sequence divergence. Furthermore, the toxigenic V. parahaemolyticus strain isolated from environments may be the potential pathogen to the local prevalent and sporadic cases.

Objective: To study the molecular epidemiology of Vibrio parahaemolyticus isolates from clinical and environmental samples collected in Hangzhou area during 2000 and 2002., Methods: V. parahaemolyticus isolates from food-poisoning, sporadic diarrhea patients and seafood in market in Hangzhou during 2000 and 2002 were serotyped. From clinical isolates of serotype O3:K6 and environmental isolates of serotype O3:KUT, virulence genes, tdh and trh, were examined by PCR. Molecular typings [including ribotyping, random amplified polymorphic DNA analysis (RAPD), enterobacterial repetitive intergenic consensus PCR (ERIC-PCR)], were also performed., Results: Among 13 food-poisoning outbreaks caused by V. parahaemolyticus, O3:K6 strains with tdh positive and trh negative were detected in 11 episodes (84.6%), and O4:K8 strains with tdh positive and trh negative were responsible for other 2 outbreaks (15.4%). In 34 V. parahaemolyticus isolates from sporadic diarrhea patients, O3:K6, O4:K8, and O1:KUT strains with tdh positive and trh negative were detected with proportions of 26.5% (9/34), 17.6% (6/34), and 38.2% (13/34), respectively, and other 6 strains were not able to be serotyped. Of 64 isolates from seafood in which both tdh and trh were negative except trh was positive in one O1:KUT strain, 37 belonged to 7 serotypes except O3:K6 or O4:K8, and 9 were not able to be serotyped. The fingprintings of ribotyping, ERIC-PCR, and RAPD showed that almost all of O3:K6 isolates from food-poisoning and sporadic diarrhea patients were genetically close to each other and there were obviously genetical difference between O3:K6 isolates from clinic and O3:KUT isolates from environment., Conclusion: There was a distinct serotype distribution between V. parahaemolyticus isolates from clinic patients and seafood. One group of close related V. parahaemolyticus O3:K6 strains with tdh positive and trh negative seemed to be prevailing in Hangzhou in those years.

Objective: To detect the RNA of severe acute respiratory syndrome virus (SARS-CoV) by using reverse transcription polymerase chain reaction (RT-PCR) targeted for a two loci and a modified nested real-time RT-PCR as to improving the reliability and sensitivity of tests., Methods: A nested RT-PCR was used for detecting one fragment of SARS-CoV RNA in oropharyngeal swabs from 3 SARS probable patients, 4 SARS suspect patients and other 27 patients with fever in Hangzhou, and the nested RT-PCR product from one SARS probable patient was sequenced. Meanwhile in these 3 SARS probable patients, other three RT-PCR methods, including a hemi-nested RT-PCR targeted for another fragment of SARS-CoV RNA, a real-time RT-PCR and a modified nested real-time RT-PCR, were employed to detect SARS-CoV RNA., Results: Two positives were found in the 3 SARS probable patients, and none positive in 4 SARS suspect patients and other 27 patients with fever, using the nested RT-PCR. The sequence of the nested RT-PCR product from one SARS probable patient was identified with the counterpart of SARS-CoV genomes published in public database. The results of the hemi-nested RT-PCR, the real-time RT-PCR and the modified nested real-time RT-PCR in the 3 SARS patients were consistent with the one of the nested RT-PCR. During detecting specimen with low copies of RNA, a weak positive signal was produced after about 35 cycles in the real-time RT-PCR, but a strong positive signal was found only after 10 cycles in the modified nested real-time RT-PCR., Conclusion: It might improve the reliability of test by employing RT-PCR targeted for two or more fragments in SARS-CoV genome. The modified nested real-time RT-PCR might have higher sensitivity than the routine real-time RT-PCR.