一种基于PCR技术的沙门氏菌血清分型试剂盒与传统血清分型方法比较

目的 评估一种基于PCR技术的沙门氏菌快速血清分型试剂盒效用,并与传统血清学分型鉴定方法进行对比,为研究人员提供一种更高效、简便的沙门氏菌血清学鉴定分型方法。方法 从本实验室菌株库中选取59株沙门氏菌,分别采用沙门氏菌血清分型PCR试剂盒和传统血清凝集法对所有菌株进行分型鉴定,比对两种分型方法的符合率。结果 59株沙门氏菌均通过传统血清凝集方法完成分型,分型率100%。59株菌株均通过沙门氏菌血清分型PCR试剂盒成功分型,分型率100%,两种方法鉴定沙门血清型符合率100%。结论 沙门氏菌血清分型PCR试剂盒能达到较高的分型率,大大缩短检测时间,减少常规血清分型的工作量及昂贵的血清消耗,节约成本,可以作为一种沙门氏菌快速血清分型方法进行应用。     

Salmonella in slaughter pigs: the effect of logistic slaughter procedures of pigs on the prevalence of Salmonella in pork

A substantial part of the finishing pigs in the Netherlands is infected with Salmonella. Infection of pigs with Salmonella can occur already on the farm. Pigs can also get infected or contaminated during transport, lairage or slaughter. The aim of this study was to evaluate the effect of separating pigs from Salmonella-infected farms from pigs from Salmonella-free farms during transport, lairage and slaughter on the prevalence of Salmonella on pork after slaughter. Two experiments were carried out. In the first experiment, farms were selected to participate, based on serology of the pigs (Dutch Salmonella ELISA). The pigs were slaughtered at the beginning of the day: firstly, sero-negative herds, secondly, sero-positive herds and thirdly, again sero-negative herds. The latter were slaughtered to investigate the effect of a contaminated slaughterline due to a previously slaughtered positive herd. In the second experiment, farms were selected to participate, based on both serology and bacteriology of the pigs on the farm. Two hundred pigs from Salmonella-free farms were slaughtered after 200 pigs from Salmonella-infected farms. Results showed that the prevalence of Salmonella in pork samples of sero-negative herds was lower than in samples of sero-positive herds. Results also showed that Salmonella contamination of carcasses after slaughter was partially caused by Salmonella-infected herds that were slaughtered before, and partially by residential flora of the slaughterhouse. It is concluded that separate slaughter of sero-negative pig herds can be useful to decrease the prevalence of Salmonella-contaminated pork after slaughter. To avoid cross-contamination by residential flora from trucks, lairage and slaughterline, cleaning and disinfection have to be improved.     

食品及临床样本中沙门菌多重耐药相关基因的研究

目的 了解从食品及临床样本中分离的沙门菌整合子以及产超广谱β-内酰胺酶(ESBL)菌分布,以及不同耐药基因与多重耐药之间的关系。方法 利用K-B法进行产ESBL菌表型确证试验;通过聚合酶链式反应方法,对产超广谱β-内酰胺酶沙门菌相关耐药基因(bla_TEM、bla_SHV、bla_CTX)以及可移动元件—整合子基因进行扩增,对整合子可变区扩增产物进行基因测序,分析携带耐药基因盒。结果 共分离309株沙门菌,138株来自食品(禽类96株,生猪肉19株,水产品23株),171株来自临床样本。309株沙门菌耐药率达78.3%,多重耐药率达41.1%,其中禽类耐药率占比最高。共检出56株产ESBL菌,其中35株携带产ESBL菌相关耐药基因(bla _TEM基因型15株,bla_CTX基因型10株,bla _TEM与bla _CTX双基因型10株),未检出bla _SHV基因。检出98株Ⅰ类整合子阳性菌,阳性率为31.7%。其中54株携带...     

食品沙门氏菌现行国内外标准中选择性增菌和分离的等效性评估

目的评估5种现行国内与国际食品沙门氏菌标准中选择性增菌步骤的等效性。方法按照GB4789.28—2013的方法使用沙门氏菌参比菌株验证5种沙门氏菌选择性增菌液亚硒酸盐胱氨酸增菌液（SC）、四硫磺酸钠煌绿增菌液（TTB）、罗伯特增菌液（RV）、穆勒-考夫曼四硫酸盐新生霉素增菌液（MKTTn）、亚硒酸盐煌绿增菌液（SBG）和5种选择性琼脂平板沙门氏菌显色琼脂平板（CAS）、木糖-赖氨酸-脱氧胆酸盐琼脂平板（XLD）、木糖赖氨酸十四烷基硫酸钠琼脂平板（XLT4）、亚硫酸铋琼脂平板（BS）和赫氏肠道菌琼脂平板（HE）的生长率（PR）和特征性生长;按照ISO 16140—2:2016和传统统计学方法设计路径,经高污染样品检测比较5种选择性增菌液和2种选择性琼脂平板的组合方法的敏感性、相对真值和可接受限值,评估10种增菌分离组合的分离敏感性、特异性、阳性预测值和阴性预测值。结果 SC对猪霍乱和猪霍乱沙门氏菌孔成道夫变种存在无效增殖;CAS和XLD识别伤寒和非伤寒、硫化氢差异表达沙门氏菌能力明显优于XLT4、HE、BS;84件样品（畜肉22件、禽肉32件和水产品30件）经5种增菌组合确认25件阳性（...     

2015—2021年湖州市食品中沙门菌污染与分子分型研究

目的 了解湖州市市售食品中沙门菌的污染状况,为预防与控制食源性疾病的发生提供依据。方法 采集2015—2021年5类1 463份样品按照GB 4789.4—2016《食品安全国家标准食品微生物学检验沙门氏菌检验》对沙门菌进行检验,对分离到的沙门菌进行血清分型、抗生素敏感试验和脉冲场凝胶电泳分子分型（PFGE）检测,结果用Excel、SPSS 19.0进行统计分析。结果 2015—2021年共检测5类1 463份食品样品,检出沙门菌菌株47株,总检出率为3.21%(47/1 463)。不同食品类别中生畜肉检出率最高,为6.61%(23/348)。共检出19种血清型的沙门菌,其中优势血清型为鼠伤寒沙门菌。不同食品中检出的沙门菌血清型有差别。对2019—2021年分离到的20株沙门菌进行药敏试验和PFGE检测,结果显示分离株对氨苄西林和四环素耐药性较强,耐药率分别为70%(14/20)和60%(12/20)。分子分型显示,经Xba I酶切后,19株沙门菌产生11个PFGE带型,多态性较高。结论 2015—2021年湖州市市售5类食品中均有检出沙门菌,其中两类为即食食品（中式凉拌菜和散装熟肉制...     

蒲公英脂溶性成分对沙门氏菌抑菌作用机理

目的:研究蒲公英脂溶性成分对沙门氏菌的抑菌活性及其作用机理。方法:采用索氏提取法提取蒲公英脂溶性成分,纸片抑菌法分析蒲公英脂溶性成分对常见食源性致病菌沙门氏菌的抑菌作用,紫外分光光度法研究了蒲公英脂溶性成分的最低抑菌浓度,通过紫外核酸物质测定、倒置荧光显微镜染色观察、电子显微镜细菌形态分析细胞膜完整性。结果:当蒲公英脂溶性成分浓度为10 mg/mL时,抑菌圈直径为11.3 mm;紫外分光光度测定最小抑菌浓度为8 mg/mL;紫外核酸物质测定、荧光染色观察及电镜细胞形态分析均显示蒲公英脂溶性成分对细胞膜有明显破坏作用。结论:蒲公英脂溶性成分对沙门氏菌有很好抑菌活性,初步确定其抑菌作用与对菌体细胞膜的破坏作用有关。     

Comparison of antimicrobial resistance in Salmonella and Campylobacter isolated from turkeys in the Midwest USA

The current study was carried out to determine the antimicrobial resistance profiles and evaluate some molecular characteristics of a set of Salmonella and Campylobacter isolates recovered from production line turkeys in the Midwest region of the United States. A total of 94 birds identified as being positive for both Salmonella and Campylobacter spp. were selected for study. All Salmonella isolates were examined for antimicrobial resistance using the methods employed in the National Antimicrobial Resistance Monitoring System (NARMS). Campylobacter isolates were subjected to similar analysis using the Etest^®. In addition, polymerase chain reaction (PCR) was carried out to determine the presence of the antimicrobial resistance associated genes, integrase (int1), class 1 integrons (Salmonella and Campylobacter) and a multidrug efflux pump (Campylobacter spp.). Results from the study showed that the Salmonella and Campylobacter isolates examined displayed resistance to a number of antimicrobials, with Salmonella and Campylobacter isolates being resistant to at least three antimicrobials while some isolates showed resistances to 6 or 8 different antimicrobials. In addition, 68.1% of the Salmonella isolates tested were found to be positive for the class I integrase gene (int1), 28.7% possessed a 1000 bp gene cassette and 17% possessed an 800 bp gene cassette. All Campylobacter isolates were negative for int1, but 36.2% tested positive for the Campylobacter multidrug efflux pump (CmeB). A considerable number of Salmonella and Campylobacter isolates tested displayed varying degrees of antimicrobial resistance as well as the presence of some factors associated with the carriage and persistence of antimicrobial resistance. Similarities in the types of antimicrobial resistance observed in Campylobacter and Salmonella strains was evident. The results of this study suggest that prescribing practice at the farm level may be a factor in promoting antimicrobial resistance in more than one species of organism. Such practices may, therefore, contribute to the potential health risk for consumers should micro-organisms carrying multiple antimicrobial resistances enter the food chain. This study may be one of the first to report on the incidence of the multidrug efflux pump (CmeB) in Campylobacters recovered from processed turkeys. The antimicrobial resistance and molecular characteristics of Salmonella and Campylobacter is discussed.     

快速检测技术在食源性沙门氏菌检测中的应用研究进展

沙门氏菌是一种广泛存在于自然界中的食源性致病菌,能导致人食物中毒,引发胃肠炎、败血症等疾病,严重时危及生命。建立快速、准确的沙门氏菌检测方法是预防和控制沙门氏菌疾病的关键,发展快速灵敏的检测方法对于保障食品质量与安全具有重要意义。本文通过对免疫学检测技术、分子生物学技术和生物传感器检测技术等在食源性沙门氏菌的检测方面的应用进行综合分析,阐明各种食源性沙门氏菌快速检测技术的原理、优缺点及研究进展,为优化食源性沙门氏菌快速检测方法提供参考。     

沙门氏菌及副溶血性弧菌的共增菌培养基的研制

为研制一种可同时富集沙门氏菌和副溶血性弧菌的增菌培养基,参照GB 4789.4-2016及GB 4789.7-2013规定的增菌培养基成分,并根据目标菌不同的营养需求,筛选适宜抑制剂和促进剂,进行单因素实验,确定共增菌培养基的配方,并验证该培养基的增菌效果。结果表明:研制出用于沙门氏菌和副溶血性弧菌的共增菌培养基成分为:蛋白胨10.0 g,磷酸二氢钾1.5 g,氯化钠7.5 g,硫代硫酸钠5.0 g,牛胆盐0.1 g,柠檬酸钠2.5 g,甘露醇2.5 g,葡萄糖2.5 g,蒸馏水1000 mL。目标菌初始接种量为10² CFU/mL,在37 ℃下培养16 h后,两种目标菌的菌浓度可达到10⁷~10⁸ CFU/mL。结果表明,共增菌培养基可用于沙门氏菌和副溶血性弧菌的富集培养,培养基配制简易,节约成本,具有良好的市场前景。     

牛肉中沙门氏菌热失活模型的建立

为了建立牛肉中沙门氏菌的热失活动力学模型,将4种不同血清型的沙门氏菌混合菌液接种到牛肉表面,将接种后的牛肉分别在55、57.5、60、62.5和65 ℃下进行加热处理。不同温度下的沙门氏菌热失活曲线用Weibull模型进行了拟合,判定系数(R²)分别为0.993(55 ℃)、0.984(57.5 ℃)、0.999(60 ℃)、0.999(62.5 ℃)和0.998(65 ℃)。进一步建立了温度对Weibull一级失活模型参数(b)影响的二级模型,即ln(b)=0.47T-28.07。用58.5和64 ℃下实际的沙门氏菌存活数对所建的模型进行验证,准确度(A_f)和偏差度(B_f)分别为1.071和1.056,0.998和1.002,均在可接受范围内。本研究所建立的模型能较好的模拟不同温度(55~65 ℃)对牛肉中沙门氏菌热失活的影响。     

2006—2021年温州地区沙门菌感染流行病学特征分析

目的 分析2006—2021年温州地区沙门菌感染流行病学特征,为沙门菌感染的防控提供科学依据。方法 对沙门菌进行鉴定确认和血清学分型,并对流行病学特征进行深入分析。结果 2006—2021年在温州地区42 300份食源性疾病腹泻患者中,共分离出沙门菌1 232株,总分离率为2.91%,共鉴定出90个血清型（14个血清群）,其中菌群以O：4（B）、O：9（D1）、O：3,10（E1）、O：8（C2-C3）、O：7（C1）为主;血清型以鼠伤寒沙门菌、肠炎沙门菌、伦敦沙门菌和德尔卑沙门菌为主;18～60岁、61岁以上和小于２岁婴幼儿是感染的主要人群;春、夏、秋季易感,具有明显的季节性;5～11月是食源性沙门菌检出的高峰期。结论 温州地区沙门菌感染在季节分布、人群分布等方面具有明显的流行病学特征,应该继续加强食源性疾病监测,进一步提高食品安全隐患的早期识别、预警与防控能力。     

A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk

Intoxications and infections caused by food-borne pathogens represent an increasing public health problem, and diagnostic tests in multiplex format are needed for the rapid identification of food contaminations caused by more than one microbial species. We have developed a multiple PCR-based platform for the simultaneous detection of the widespread milk-associated pathogens Salmonella spp., Listeria monocytogenes and Escherichia coli O157. The assay combines an enrichment step in a medium properly formulated for the simultaneous growth of target pathogens, a DNA isolation method, and a multiplex Real-Time PCR detection system based either on dual-labelled probes (mRT-PCR), or on melting curve analysis (mHRM). The second, producing a distinct peak for each amplification product, allows the qualitative assessment of pathogen presence. Moreover, the internal amplification control (IAC) included in the reaction, ensuring the reliability of results, complies with quality management programmes. Inclusivity and exclusivity were 100% each, with a detection limit of 1 CFU for each pathogen in a total of five 25 ml-aliquots of raw milk, and a duration of two working days.The assay represents an alternative approach for the qualitative detection of the cited bacterial species, suitable for a relatively inexpensive screening of several milk samples, reducing the turnaround time and the workload.     

2021年广西腹泻病人来源的鼠伤寒沙门菌及其单相变种比较分析

目的 通过研究比较2021年广西壮族自治区腹泻病人来源的鼠伤寒沙门菌及其单相变体1，4，［5］，12∶i∶-（S.1，4，［5］，12∶i∶-）流行特征与耐药情况，更好地了解鼠伤寒沙门菌及其单相变体多重耐药克隆的流行病学及其在传播的潜力。方法 对来自病例监测分离的276株鼠伤寒沙门菌用血清凝集方法进行第一次分型，当第二项鞭毛抗原诱导三次后依然不凝集，再用多重PCR的方法进行第二次分型，用微量肉汤稀释法进行药敏试验。结果 经多重PCR确认为鼠伤寒沙门菌单相变体的有201株（72.8%），鼠伤寒沙门菌75株。鼠伤寒沙门菌对磺胺类药物、氯霉素的耐药率显著高于鼠伤寒沙门菌单相变体（P<0.05）。鼠伤寒沙门菌单相变体对氨苄西林、头孢噻肟、萘啶酸、庆大霉素、四环素5种抗生素的耐药率显著高于鼠伤寒沙门菌（P<0.05）。两种沙门菌对3类及以上抗生素耐药率达到79%以上，共同多重优势耐药谱为耐氨苄西林、氯霉素、甲氧苄啶/磺胺甲唑和四环素。结论 鼠伤寒沙门菌单相变体已经超过鼠伤寒沙门菌成为广西壮族自治区腹泻病人中最优势的血清型。两种沙门菌耐药情况不容乐观，特别是多重耐药菌株的快速增加。需要有针对性地加强对食源性鼠伤寒沙门菌和鼠伤寒沙门菌单相变体进行耐药监测，揭示其耐药性的潜在决定因素，并展开有效的干预措施。     

奶粉中沙门菌的检测能力验证研究

目的 通过组织实施奶粉中沙门菌的能力验证,对国内实验室的沙门菌检测能力进行分析和评价。方法 采用人工污染的方式制备奶粉中沙门菌能力验证样品,即菌种复苏、增菌后配制成菌悬液加入配制好的复原牛乳中混匀分装,进行冷冻干燥;对制备好的样品进行均匀性检验和稳定性检验。本次能力验证设计3组不同的能力验证样品,每个实验室随机发送2个样品,将各参加实验室提交的检测结果与指定值比较,评定各参加实验室的测试结果为满意或不满意（假阴性或假阳性）。结果 全国共337家实验室参加该项沙门菌的检测能力验证,有满意结果的实验室323家,满意率为95.8%,有不满意（假阴性和假阳性）结果的实验室14家,占比为4.2%。共向实验室发送674个样品,658个结果满意,满意率为97.6%;16个样品结果不满意（假阴性或假阳性）,占比为2.4%。结论 采用不同组别的样品随机编号且随机发样的方式,取得了良好的考核效果;本次人工污染方式制备的能力验证样品,充分考虑了目标菌、类似干扰菌和背景菌,制备的样品与实际样品一致性较好,考核效果良好。能力验证评价结果表明,全国参加考核的实验室奶粉中沙门菌检测能力总体较好,少部分实验室的检测能...     

PCR-based fingerprinting techniques for rapid detection of animal species in meat products

A reproducible, rapid, and simple method for simultaneous identification of multiple meat species in a single step DNA-based test has been developed based on the generation of species-specific fingerprintings by two different arbitrary DNA amplification approaches (RAPD- and AP-PCR). Samples representative of various species and meat products submitted to different processing conditions were selected to verify the applicability of the techniques. RAPD-PCR fingerprintings allowed the discrimination amongst pork, beef, lamb, chicken and turkey in all cases. Samples corresponding to each species were clustered together at similarity levels ?75%. The DNA profiles consisted of a discrete but reproducible number of bands, which made possible the interpretation of the results by simple visual inspection. AP-PCR also allowed identification of the five tested species in every sample although more complex patterns were generated, including some low intensity bands. In both cases, a ramp time between annealing and extension temperatures was introduced to achieve good reproducibility. Overall, the simplicity of RAPD-PCR patterns could make this technique suitable for meat authentication in routine analysis.     

Antimicrobial resistant genes associated with Salmonella from retail meats and street foods

We examined the antimicrobial resistance of Salmonella isolates from 300 meat products (raw beef, chicken meat and street foods). A total of 88 non-duplicate Salmonella from 66 (22.0%) retail meat and 22 (7.5%) street food samples were recovered and 11 serovars were identified. Among the 88 Salmonella isolates, the highest resistance was to tetracycline (73.8%), followed by sulfonamide (63.6%), streptomycin (57.9%), nalidixic acid (44.3%), trimethoprim–sulfamethoxazole (19.3%), ampicillin (17.0%), chloramphenicol (10.2%), cephalotin (8.0%), kanamycin (6.8%), ciprofloxacin (2.2%) gentamycin (2.2%), cefoxitin (2.2%), amoxicillin–clavulanate (1.0%) and amikacin (1.0%). Sixty-seven percent of the isolates (59/88) were multidrug resistant (MDR). Ten out of 17 resistance genes (bla_TEM-1, strA, strB, aadA, sulI, sulII, tetA, tetB, floR, cmlA) were detected. Twelve of the 59 MDR Salmonella isolates from serovars Typhimurium (6), Newport (3), Agona (1), Albany (1) and Weltevreden (1) had class 1 integrons. The gene cassettes identified were dfrA1, dfrV, dfrA12, aadA2, sul1 genes and an open reading frame orfC of unknown function. Four integron-positive isolates could transfer resistance phenotypes to the recipient strain, E. coli J53 via conjugation. These data revealed that the Salmonella isolates recovered from the retail meats and cooked street foods were resistant to multiple antimicrobials, which can be transmitted to humans through food products. The occurrence of mobile genetic elements such as integrons reiterates the roles of food of animal origins as a reservoir of MDR Salmonella.     

赣州市食源性腹泻患者分离沙门菌耐药性和分子分型特征研究

目的 了解赣州市食源性沙门菌耐药性和分子分型特征,建立赣州市食源性沙门菌耐药性和分子指纹图谱数据库,为临床合理用药和食源性沙门菌病的暴发溯源提供科学依据。方法 对赣州市2020—2022年食源性疾病主动监测中分离的136株沙门菌进行血清分型、药物敏感性试验、全基因组测序（WGS）和脉冲场凝胶电泳（PFGE）,并进行耐药基因注释和图谱聚类分析。结果 赣州市食源性沙门菌对STR耐药率最高（83.09%）,其次为TET（78.68%）和AMP（76.47%）;多重耐药菌株占76.47%,耐药谱型广泛,主要流行耐药谱型为AMP-TET-CHL-STR-SXT;WGS预测出7种类别共61种耐药基因,以氨基糖苷类耐药基因携带率（99.19%）最高,大环内酯类（8.87%）最低;136株沙门菌以鼠伤寒变种和鼠伤寒为优势血清型,经PFGE分子分型分为98种带型。结论 赣州市食源性沙门菌耐药状况严重,耐药基因携带率高且基因型多样,PFGE分子型别呈多态性,优势血清型别可能引起暴发流行,应加强监测和研究。     

福建省1975—2021年沙门菌血清型分布和表型特征

目的 回顾分析1975—2021年福建省人源和非人源沙门菌的血清型和表型特征。方法 收集1975—2021年福建省内临床、健康从业者、流通生鲜食品和环境中沙门菌株,使用世界卫生组织推荐沙门菌血清分型方法,基于全基因组测序的两种预测软件比对血清型及变种,使用生化组合和飞行质谱辅助鉴定同抗原式、不同菌型或表型变异菌型。结果 4 693株人源和非人源沙门菌分布于含4个亚种的21个血清群（病人源18个、健康携带源9个、食品源17个、环境源8个）;甲型副伤寒、伤寒、肠炎、猪霍乱、鼠伤寒、罗森和斯坦利是30个肠外侵袭血清型中的优势型;除伤寒、甲型副伤寒和乙型副伤寒外的沙门菌血清型有96个（病人源63个、健康携带源51个、食品源60个、环境源20个）,临床前10位菌型为鼠伤寒、伤寒、肠炎、德比、斯坦利、甲型副伤寒、鸭、韦太夫雷登、罗森、伦敦;食品源前10位菌型为鸭、德比、鼠伤寒、斯坦利、韦太夫雷登、纽波特、肠炎、汤卜逊、阿贡纳、伦敦;确认14个血清型（包括10个双相菌和4个单相菌）存在单相变种;证实多个血清型存在硫化氢阴性、发酵乳糖或蔗糖、菌落黏液型和产色素型等表型变异现象。结论 福建省沙门菌血清型...     

Evaluation of a rapid PCR-based method for the detection of animal material

A rapid PCR-based analytical method for detection of animal-derived materials in complete feed was developed. Using a commercially available DNA forensic kit for the extraction of DNA from animal feed, a sensitive method was developed that was capable of detecting as little as 0.03% bovine meat and bone meal in complete feed in under 8 h of total assay time. The reduction in assay time was accomplished by reducing the DNA extraction time to 2 h and using the simpler cleanup procedure of the kit. Assay sensitivity can be increased to 0.006% by increasing the DNA extraction time to an overnight incubation of approximately 16 h. Examination of dairy feed samples containing either bovine meat and bone meal, porcine meat and bone meal, or lamb meal at a level of 0.1% (wt/wt basis) suggested that this method may be suitable for regulatory uses. The adoption of this commercially available kit for use with animal feeds yields an assay that is quicker and simpler to perform than a previously validated assay for the detection of animal proteins in animal feed.