四川省肉毒梭菌PCR基因分型方法比较研究

目的比较分析4种肉毒毒素(botulinum neurotoxins,Bo NT)基因分型方法,为四川省监测和食物中毒中肉毒梭菌(Clostridium botulinum)分型鉴定提供可靠的方法。方法使用本实验室保存的6株肉毒梭菌(包括A、B、E型)验证4种肉毒梭菌PCR基因分型鉴定方法——美国食品药品监督管理局(FDA)多重PCR分型方法、国际标准化组织(ISO)的两种多重PCR分型方法和一种实时荧光PCR分型方法,比较方法间的差异,并初步分析差异原因。结果 3种多重PCR方法均可在一个反应中同时检测A、B、E 3种型别肉毒梭菌。ISO多重PCR方法 1中A型检测虽能获得预期条带,但结果条带不清晰。其余两种多重PCR方法在分型检测肉毒梭菌时,可获得清晰的预期条带。实时荧光PCR分型方法能在多重反应体系中同时检测到不同型的肉毒梭菌,但由于荧光标记相同,要获得分型结果需要分别检测各毒素型别。结论美国FDA多重PCR方法和ISO多重PCR方法 2操作较简单易行,可在四川省肉毒梭菌监测中推荐使用。     

新疆豆制品生产环境中肉毒梭菌的分离鉴定

从新疆伊犁察布查尔县的土壤中分离到1株优势菌,该分离株生长特性与A型肉毒梭菌(Clostridiumbotulinum type A)一致,为革兰氏阳性粗大杆菌,在EYA培养基上菌落边缘呈锯齿状,表面粗糙不规则,有较大的乳浊环。根据分离株的形态、生理生化特性,结合琼脂糖凝胶凝胶电泳图像、16SrDNA V6-V8序列(GenBank登录号:JN248616)与系统发育树分析,将其鉴定为A型肉毒梭菌。     

红曲色素、乳酸链球菌素、山梨酸钾对肉毒梭状芽孢杆菌的抑制研究

试验对红曲色素、乳酸链球菌素和山梨酸钾三种物质抑制肉毒梭状芽孢杆菌的情况与ＮａＮＯ＿２抑制肉毒梭状芽孢杆菌进行了比较，证明三种物质都具有不同程度的抑制肉毒梭菌生长的能力。Ｎｉｓｉｎ和山梨酸钾的抑制作用较红曲色素更强。     

Shelf Life and Clostridium botulinum Toxin Development during Storage of Modified Atmosphere-packaged Fresh Catfish Fillets

Shelf life (onset of sensory spoilage) and potential for toxin production by Clostridium botulinum type E in retail type packages of fresh catfish fillets in high barrier film were investigated under selected atmospheres when stored under refrigeration and temperature-abuse conditions. Shelf life of fillets in all atmospheres decreased with increase of storage temperature from 4°C to 16°C. Trimethylamine content associated with onset of spoilage was different for each storage temperature and atmosphere. Surface pH and K-values were not good indicators of onset of sensory spoilage. Toxin development coincided with sensory spoilage at 16°C storage for fillets packaged in either atmosphere. At 4°C, none of the MA-packaged fillets became toxic, even after 37 days of sensory spoilage.     

Germination, Growth, and Toxin Production of Nonproteolytic Clostridium botulinum as Affected by Multiple Barriers

ABSTRACT The effect of combinations of pH (6.5, 5.75), NaCl (0.25,1.75%), and incubation temperatures (7,13 °C) on spore germination, outgrowth, and time to toxicity of nonproteolytic Clostridium botulinum was examined in a broth system. Spores of four toxin type E and nonproteolytic type B were inoculated (10⁴/ml) into Tryptone Peptone Glucose Yeast Extract (TPGY) broth. Cultures were monitored for three weeks, or until toxin was detected. A modified FSIS‐amplified ELISA (comparable in sensitivity to the mouse bioassay) was used to screen cultures for neurotoxin. Combinations of the most inhibitory level for each barrier reduced the degree and rate of germination, the lag and growth rate of vegetative cells, and the time to toxicity.     

Theoretical comparison of a new and the traditional method to calculate Clostridium botulinum survival during thermal inactivation

When published isothermal survival data of Clostridium botulinum spores in the range 101–121 °C were plotted in the form of logS(t) vs t relationships, where S(t) is the momentary survival ratio, they were all non‐linear. They had a noticeable upward concavity, in violation of the assumption that sporal inactivation is a process that follows first‐order reaction order kinetics. They could be described by the power law model logS(t) = ? b(T)t ^n(T), where b(T) and n(T) are temperature‐dependent coefficients of the order of 0.1–6 and about 0.4 respectively. These coefficients were used to construct simulated survival curves under different heating regimes with a recently proposed model. The model is based on the assumption that the local slope of the non‐isothermal survival curve, or the momentary inactivation rate, is determined solely by the momentary temperature and survival ratio, which in turn are functions of the population thermal history. The survival curves calculated with this model differ considerably from those produced by the standard method based on the traditional D and Z values. The shortcomings of the standard model are that these values depend on the number of points taken for the regression, and that its predicted survival ratios depend on the selected reference temperature. The differential equation which is proposed to replace it can be solved numerically using a program such as Mathematica®. Its predictions solely depend on the observed survival patterns under isothermal conditions and not on any preconceived kinetic model. Nevertheless, the method still needs verification with experimental non‐isothermal survival data, as has already been done with Listeria and Salmonella cells. © 2001 Society of Chemical Industry     

Nisin-resistant (Nisr) Listeria monocytogenes and Nisr Clostridium botulinum Are Not Resistant to Common Food Preservatives

Nisin‐resistant (Nisr) strains of Clostridium botulinum and Listeria monocytogenes may arise as nisin becomes more widely used as an additional safety barrier in minimally‐processed foods. The sensitivity of Nisr L. monocytogenes ATCC 700301 and ATCC 700302 and toxigenic Nisr C. botulinum 169B to low pH, salt, sodium nitrite, and potassium sorbate was assayed using discontinuous gradients in broth and compared to the parental wild‐type strains. The nisin‐resistant strains did not have intrinsic resistance to low pH, sodium chloride, potassium sorbate, or sodium nitrite. In no case were the Nisr L. monocytogenes and C. botulinum strains examined more resistant to inhibitors than the parental strains.     

某企业婴儿肉毒中毒乳粉相关样品中肉毒梭菌的分离与分型

目的 对从某企业获取的30批次婴儿配方乳粉样品进行肉毒毒素和肉毒梭菌检测,对分离到的1株B型肉毒梭菌进行全基因组测序分析。方法 参照GB 4789.12—2016《食品安全国家标准 食品微生物学检验 肉毒梭菌及肉毒毒素检验》对样品进行肉毒梭菌分离及肉毒毒素分型实验;对分离到的菌株进行全基因组测序,并分析菌株的遗传特征。结果 30批次样品中均未检出肉毒毒素;将增菌液进行小鼠腹腔注射后,4批次乳粉样品出现了典型小鼠肉毒中毒症状,但仅从1批次乳粉样品中分离到肉毒梭菌。基因组测序分析显示,该菌为Ⅰ群B型肉毒梭菌,毒素基因簇为Ha型,毒素基因为B2亚型。结论 针对背景微生物复杂的婴儿配方乳粉中肉毒梭菌检测,不应以菌种分离作为金标准,而应以增菌液小鼠毒性实验结合肉毒抗血清保护实验作为确认方法。全基因组测序可对分离菌种进行精准鉴定和相关遗传特征分析,为中毒事件处理提供可靠的技术支撑。     

Prevention of Clostridium botulinum Type A, Proteolytic B and E Toxin Formation in Refrigerated Pea Soup by Lactobacillus plantarum ATCC 8014

The ability of Lactobacillus plantarum ATCC 8014 to inhibit Clostridium botulinum toxin production in pea soup was investigated. Soup containing C. botulinum spores (10³/g) with and without L. plantarum (10⁶/g) were evaluated. Soup containing only type A spores was toxic on days 1 and 2 when incubated at 35°C and 25°C, respectively. Soup containing only proteolytic type B spores was toxic on days 2 and 5 at 35°C and 25°C, respectively. Soup containing only type E spores was toxic at 25°C, 15°C, and 5°C in 7, 7, and 63 days respectively. No toxin was found in soup containing C. botulinum spores plus L. plantarum at any temperature studied.     

Development and validation of a real-time PCR assay specific for Clostridium estertheticum and C. estertheticum-like psychrotolerant bacteria

A new real-time PCR assay was developed targeted to the psychrotolerant spoilage bacteria, Clostridum estertheticum, a causative agent of 'blown-pack' spoilage of vacuum packaged meats during chilled storage. Further, a robust validation of the sensitivity and specificity in different meat processing related matrices was carried out. Results show that real-time PCR is a valid method for the detection of C. estertheticum spores as long as consideration is given to the matrix being tested and the sensitivity of detection required. For meat, hide, blood/drip and environmental swabs it was possible to detect low numbers of C. estertheticum spores (approx 3 spores per ml) by direct real-time PCR (without pre-enrichment of the samples). For faeces and soil matrices, a cold temperature enrichment step was required prior to DNA extraction and real-time PCR analysis to increase the ability to detect samples containing C. estertheticum spores; this was particularly important when the samples contained low numbers of spores (less than 3 spores per ml). For matrices with high levels of PCR inhibitors such as soil, it was necessary to dilute the extracted DNA sample 100 fold especially for detection of high levels of contamination (greater than 10(3)per ml) otherwise a pre-enrichment was required.     

Differentiation of group I and group II strains of Clostridium botulinum by focal plane array Fourier transform infrared spectroscopy

A method was developed for whole-organism fingerprinting of Clostridium botulinum isolates by focal plane array Fourier transform infrared (FPA-FTIR) spectroscopy. A database of 150,000 infrared spectra of 44 strains of C. botulinum was acquired using a FPA-FTIR imaging spectrometer equipped with a 16 x 16 array detector to evaluate the ability of FTIR spectroscopy to differentiate the 44 strains. The database contained strains from C. botulinum groups I and II producing botulinum neurotoxin of serotypes A, B, E, and F. All strains were grown on each of three agar media (brain heart infusion, McClung Toabe agar base, and universal) prior to spectral acquisition. Given the dependence of the infrared spectra of microorganisms on the composition of the growth medium, the spectra were initially separated into three subsets corresponding to the three growth media employed. However, the replicate spectra of all strains, regardless of growth medium, were properly clustered by hierarchical cluster analysis based on differences in their infrared spectral profiles in three narrow spectral regions (1,428 to 1,412, 1,296 to 1,284, and 1,112 to 1,100 cm(-1)). The dendrogram generated from the FTIR data revealed complete separation between group I and group II strains. The spectral differences between group I and group II strains allowed accurate classification of C. botulinum strains at the group level in two blind validation studies (n = 40). These results demonstrate that FPA-FTIR spectroscopy has the potential for rapid discrimination of group I and group II C. botulinum strains in less than 3 min per sample.     

Characterisation of Clostridium botulinum groups I and II by randomly amplified polymorphic DNA analysis and repetitive element sequence-based PCR.

Random amplified polymorphic DNA analysis (RAPD) and repetitive element sequence-based PCR (rep-PCR) were evaluated with respect to their applicability to characterise Clostridium botulinum group I and II strains, the species causing human botulism. Fifteen group I and 21 group II strains of various geographical and temporal origins were characterised with four single arbitrary RAPD primers at low stringency amplification conditions and with a degenerate REP primer pair at moderately stringent conditions. Ready-To-Go RAPD Analysis Beads and Ready-To-Go PCR Beads were used for PCR reactions with RAPD and rep-PCR, respectively. Arbitrary primer OPJ 6 yielded the most discriminating patterns, and distinguished group II C. botulinum serotypes at the strain level. Group I strains were mainly discriminated at the serotype level. The discriminatory power of rep-PCR was found to be inferior to that of RAPD. The REP1R-Dt and REP2R-Dt primer pair generated group I- and II-specific fragments and arbitrary primer OPJ 13 produced a serotype E-specific fragment. The use of pre-dispensed and pre-optimised beads attributed to highly reproducible results. As compared to more time-consuming typing methods, such as pulsed-field gel electrophoresis (PFGE), both RAPD and rep-PCR were characterised by rapid performance and a typeability of 100%.     

Development and validation of multiplex PCR assay for differentiating tunas and billfishes

Commercially available tunas and billfishes are generally processed as steaks, making it difficult to visually distinguish between the two. We developed and validated species-specific primers to prevent the adulteration of tunas by billfishes. Tunas and billfishes primers were designed on the cytochrome oxidase subunit I. Multiplex PCR bands obtained were 579 bp, 291 bp and 114 bp for tunas, billfishes and internal control. Sensitivity was determined to be 5 ng for tunas and billfishes. A total of 50 samples were monitored: 49 for tunas and 1 for billfish. As a result of the monitoring, the fake tunas did not show due to the agreement between product name and the raw material of the wrapping paper. Our results indicate that the species-specific primers developed in this study are suitable for differentiating tunas and billfishes. The newly developed multiplex PCR assay is a time and cost effective technique for determining the authenticity of tunas and billfishes.     

Hazard and control of group II (non-proteolytic) Clostridium botulinum in modern food processing

Group II (non-proteolytic) Clostridium botulinum poses a safety hazard in modern food processing, which consists of mild pasteurization treatments, anaerobic packaging, extended shelf lives and chilled storage. The high risk is reflected in the relatively large number of botulism cases due to group II C. botulinum in commercially produced foods during the past decades. Because of the high prevalence of group II C. botulinum in the environment, food raw materials may carry spores. Although group II spores are less heat-resistant than group I (proteolytic) spores, they can tolerate the heat treatments employed in the chilled food industry. Some food components may actually provide spores with protection from heat. Spore heat resistance should therefore be investigated for each food in order to determine the efficiency of industrial heat treatments. Group II strains are psychrotrophic and thus they are able to grow at refrigeration temperatures. Anaerobic packages and extended shelf lives provide C. botulinum with favourable conditions for growth and toxin formation. As the use of salt and other preservatives in these foods is limited, microbiological safety relies mainly on refrigerated storage. This sets great challenges on the production of chilled packaged foods. To ensure the safety of these foods, more than one factor should safeguard against botulinal growth and toxin production.     

A unique restriction site in the flaA gene allows rapid differentiation of group I and group II Clostridium botulinum strains by PCR-restriction fragment length polymorphism analysis

Clostridium botulinum produces the potent botulinum neurotoxin, the causative agent of botulism. Based on distinctive physiological traits, strains of C. botulinum can be divided into four groups: however, only groups I and II are associated with human illness. Alignment of the flaA gene sequences from 40 group I and 40 group II strains identified a single BsrG1 restriction cut site that was present at base pair 283 in all group II flaA sequences and was not found in any group I sequence. The flaA gene was amplified by rapid colony PCR from 22 group I strains and 18 group II strains and digested with BsrGI restriction enzyme. Standard agarose gel electrophoresis with ethidium bromide staining showed two fragments, following restriction digestion of group II flaA gene amplicons with BsrGI, but only a single band of uncut flaA from group I strains. Combining rapid colony PCR with BsrGI restriction digest of the flaA gene at 60 degrees C is a significant improvement over current methods, such as meat digestion or amplified fragment length polymorphism, as a strain can be identified as either group I or group II in under 5 h when starting with a visible plated C. botulinum colony.     

A novel 4-(2-pyridylazo) resorcinol functionalised magnetic nanosorbent for selective extraction of Cu(II) and Pb(II) ions from food and water samples

This paper describes a novel sorbent based on 4-(2-pyridylazo) resorcinol functionalised magnetic nanoparticles and its application for the extraction and pre-concentration of trace amounts of Cu(II) and Pb(II) ions. The nanosorbent was characterised by Fourier transform infrared spectroscopy, X-ray powder diffraction, thermal analysis, elemental analysis and scanning electron microscopy. The effects of various parameters such as pH, sorption time, sorbent dosage, elution time, volume and concentration of eluent were investigated. Following the sorption and elution of analytes, Cu(II) and Pb(II) ions were quantified by flame atomic absorption spectrometry. The limits of detection were 0.07 and 0.7 μg l^?1 for Cu(II) and Pb(II), respectively. The relative standard deviations of the method were less than 7%. The sorption capacity of this new sorbent were 92 and 78 mg g^?1 for Cu(II) and Pb(II), respectively. Finally this nanosorbent was applied to the rapid extraction of trace quantities of Cu(II) and Pb(II) ions in different real samples and satisfactory results were obtained.     

转基因产品中bar和pat基因的SYBR Green I-PCR检测方法研究(英文)

SYBRGreenI是一种在实时定量PCR检测中广泛使用的DNA结合染料。PCR结果可以通过溶解曲线分析来鉴定而不再需要使用传统的琼脂糖凝胶电泳来鉴定,因为PCR特定扩增产物的溶解温度的特异性是类似于琼脂糖凝胶电泳中的扩增条带的特异性。建立了基于扩增产物溶解曲线分析的二重实时定量PCR对转bar和pat基因的作物进行检测的方法。获得了bar和pat基因的特异性扩增产物和他们的特定溶解温度,并且通过几组不同的二重PCR对他们的扩增产物进行了溶解曲线鉴定。还对影响溶解曲线分析的一些因素进行了研究和讨论。结果表明基于扩增产物的溶解温度建立的二重PCR检测方法是可行的,该方法对转bar和pat基因的作物可达到0.9%的检测限。     

Effect of Rooibos (Aspalathus linearis) on Growth Control of Clostridium perfringens and Lipid Oxidation of Ready‐to‐Eat Jokbal (Pig's Trotters)

This study investigated the antimicrobial effects of rooibos (tea extract), potassium lactate (PL) and sodium diacetate (SDA) mixture alone or in combinations on the growth of Clostridium perfringens vegetative cell and spore in ready‐to‐eat (RTE) Jokbal (pig's trotters). Addition of a combination of 10% rooibos and 4% PL + SDA inhibit growth of C. perfringens vegetative cell in Jokbal at 24 °C and 36 °C. The significant inhibition on germination and growth of C. perfringens spores was also observed in Jokbal with a combination of 10% rooibos and 4% PL + SDA (PL: 2.24%, SDA: 0.16%) at 24 °C. The Jokbal treated with 10% rooibos and 4% PL + SDA mixture had significantly (P < 0.05) lower TBARS values than the control at 10 and 24 °C. The lipid oxidation inhibition effect was the highest (P < 0.05) in anaerobic packed Jokbal with 10% rooibos. The addition of a combination of 10% rooibos and 4% PL + SDA during the processing of Jokbal prevented the growth of C. perfringens and the germination and growth of C. perfringens spores at room temperature. This study shows rooibos tea as a valuable natural food preservative in meat products.