SPA-ELISA用于单核细胞增生性李斯特菌检验的应用研究

本研究创造性地将SPA-ELISA应用于食品中单核细胞增生性李斯特菌(简称LM)的检验中,建立了快速检测该菌的ELISA方法.试验表明:该方法可特异性检测肉品中的LM,样品中最低检出量为5个/10克,经增菌后的培养液最低检测限可达到105CFU/ml,并可在48小时内报告结果,比常规分离培养鉴定方法快5～6天,适于LM的快速筛选.     

传统发酵豆制品中3种致病菌的三重荧光PCR快速检测方法的建立

为了建立传统发酵豆制品中单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的三重荧光PCR快速检测方法。以单增李斯特菌hly A基因、蜡样芽孢杆菌Cereolysin AB基因和金黄色葡萄球菌nuc基因为靶基因设计引物与TaqMan探针,通过优化PCR反应体系,建立了可同时检测单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的三重荧光定量PCR体系,并进行了特异性和敏感性试验。结果显示,该方法灵敏度高,特异性强,重复性好。对26株非目标菌进行检测,结果均为阴性,而定量检测批内和批间的变异系数均小于2%。单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌敏感性试验结果表明,这三种细菌的最低检测浓度分别为3×103cfu/mL、2×104cfu/mL、2×104cfu/mL。应用该方法可在8h内完成对样品中单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的同步检测。     

单增李斯特菌胶体金免疫层析试纸条的研制

以单增李斯特菌(LM)内化素A蛋白(InlA)单克隆抗体为基础,研制其胶体金免疫层析检测试纸条。方法 采用DNAStar软件对LM inlA全长基因编码蛋白进行抗原表位分析,截取部分inlA基因片段构建原核表达质粒,诱导表达和纯化获得重组蛋白。以该蛋白免疫BALB/c小鼠,筛选高效分泌抗InlA单克隆抗体的杂交瘤细胞株,制备单克隆抗体;以双抗体夹心的原理研制胶体金免疫层析检测试纸条,并对其特异性、敏感性、稳定性进行评价。结果 筛选到2株高效分泌抗InlA单克隆抗体的杂交瘤细胞株,抗体属于IgG1亚类,小鼠腹水抗体效价为1∶64000;研制的试纸条可与LM发生阳性反应,而与非LM李斯特菌、链球菌、鼠伤寒沙门菌、大肠杆菌O157∶H7等食源性致病菌均不发生阳性反应;LM纯培养物敏感性为2.4×10^5cfu/ml,模拟猪肉样品敏感性为4.0×10⁶ cfu/ml;4℃保存期可达16周以上。结论 研制的胶体金免疫层析试纸条具有快速、特异、敏感等优点,可以用于样品中LM的快速检测。     

基因探针快速检测食品中单增李斯特氏菌

应用AccuProbe基因探针方法快速检测食品中单增李斯特氏菌(Listeriamonocytogenes),结果表明,采用该基因探针方法检测食品中单增李斯特氏菌特异性强,对食品中单增李斯特氏菌鉴定时间仅需30min,采用增菌肉汤检测低限为7.5×107cfu/mL。对206个样品检测结果表明,AccuProbe基因探针方法可快速准确地检测食品中单增李斯特氏菌。     

TaqMan探针法荧光定量PCR检测鲜切甜瓜中单核细胞增多性李斯特菌的研究

根据多条单核细胞增多性李斯特菌的hly基因序列,设计了1对简并引物和1条TaqMan探针,建立荧光定量PCR快速检测单核细胞增多性李斯特菌的方法,对该方法的特异性、灵敏度进行了验证,并采用该法对染菌的鲜切甜瓜进行了检测。结果表明,该方法具有较好的特异性,对质粒标准品的灵敏度为1.12×102copies/μL,对染菌的鲜切甜瓜中单核细胞增多性李斯特菌的最低检出限为6.28×102cfu/mL。该方法可为快速检测鲜切果蔬病原微生物污染度及其控制奠定技术基础。     

乳品中致病大肠杆菌荧光双重PCR技术建立

建立乳及乳制品中致病性大肠杆菌(EPEC)的快检方法。以EPEC特异的微绒毛粘连基因(eae gene)和茵毛束形成编码基因(bfp gene)为模板,设计两对引物,荧光探针对EPEC进行特异性扩增。该方法快速,特异性好,对EPEC的最低检出量为1 cfu/μL(纯菌),检出时间为3 h,同一样品重复检测3次ct值的变异系数均小于5%。建立可快速特异检测乳及乳制品中EPEC的双重荧光PCR方法。     

水产品生产加工环节致病菌污染的多重PCR检测

运用多重PCR分子方法对冷冻鱼丸生产加工环节中的霍乱弧菌(VC)、副溶血性弧菌(VP)和单核细胞增生李斯特氏菌(LM)进行检测,检测灵敏度为103 cfu/mL.研究过程中确定鱼丸水煮环节为关键控制点(CCP).方法能快速、灵敏地对水产品生产加工环节中的致病菌进行有效检测.     

水产品中副溶血性弧菌PMA-ddPCR活菌定量检测方法的研究

本研究旨在针对水产品中活的副溶血性弧菌建立一种快速定量的PCR检测方法。基于叠氮溴化丙锭(PMA)在一定光照条件下能抑制死亡菌DNA扩增,以及微滴式数字PCR技术能将检测精度扩展至单分子目标基因,并实现绝对定量的特点,以副溶血性弧菌tlh基因为目的片段设计及筛选适合的特异性引物与探针,优化反应体系,通过对PMA浓度及曝光条件等优化,建立了一种联用PMA-dd PCR技术快速检测水产品中活的副溶血性弧菌的定量检测方法。研究结果显示:选择16μg/m L作为PMA工作浓度,曝光时间为8 min,此条件下能够完全抑制副溶血性弧菌死菌的DNA扩增并对活菌扩增无影响。通过对比PMA-dd PCR和PMA-q PCR的检测低限分别为2×10~1 cfu/mL、2×10~2 cfu/mL,PMA-dd PCR法的灵敏度比PMA-q PCR法的高。应用PMA-dd PCR定量方法检测人工污染的基围虾和小帆立贝这两种海产品,在基围虾中最低可检出1.9×10~1 cfu/g的副溶血性弧菌、在小帆立贝中最低可检出8.9 cfu/g的副溶血性弧菌。该研究为将PCR技术实际应用于水产品中低量污染、活的副溶血性弧菌的定量检测奠定了基础。     

3种致病菌多重PCR检测体系的建立及应用

金黄葡萄球菌(Staphylococcus aureus,SA)、单核细胞增生性李斯特菌(Listeria monocytogen,LM)和蜡样芽孢杆菌(Bacillus cereus,BC)是食品中重要的致病菌,建立其多重PCR的快速检测体系,对开发食源性致病微生物快速检测试剂盒具有重要意义。根据SA的nuc基因、LM的hly基因和BC的hemolysin基因,设计合成3对特异性引物,然后进行单基因PCR反应特异性验证。在此基础上建立了3种致病菌的多重PCR检测体系,并应用于食品检测中,同时以国标法进行对比验证。结果表明,建立的多重PCR检测方法简单、快速、灵敏度高,检测灵敏度可达到1 cfu/mL,整个检测时间在16 h以内,具有较大的应用价值,可广泛应用于食品卫生检测以及临床检验等领域。     

RapidChek SELECT检测食品中沙门菌方法的评价

目的评价Rapid Chek SELECT方法对食品中沙门菌的检测效果并验证。方法用添加并回收沙门菌标准菌株方法验证Rapid Chek SELECT的检测限,添加非沙门菌标准菌株方法测定其特异性,以国标法为参比,通过检测实际样品,对Rapid Chek SELECT方法进行验证。结果 1Rapid Chek SELECT沙门菌检测试纸条的检测限为1 cfu/25 g(或1 cfu/ml);2与10种非沙门菌无交叉反应特异性;3直接检测食品中沙门菌的最低浓度为106cfu/ml;4对实际样品中沙门菌的检测结果显示,Rapid Chek SELECT方法和国标方法阳性率分别为87.5%(35/40)和85%(34/40),两种方法最终检测结果的符合率为97.5%(39/40),Rapid Chek SELECT方法等同于国标方法。结论与国标方法相比,Rapid Chek SELECT沙门菌检测试剂盒灵敏度高、特异性强、操作简便,有效减少非沙门菌的干扰、省时,适用于食品中沙门菌的快速检测。     

乳酸菌协同小麦胚芽油发酵对黑曲霉生长活性的影响

本文通过初筛和复筛考察不同乳酸菌的抗真菌活性,并将小麦胚芽油添加入乳酸菌培养液中进行发酵,旨在明确乳酸菌协同小麦胚芽油发酵对抗黑曲霉活性的影响。研究表明,小麦胚芽油不具有抑制黑曲霉生长的能力,而乳杆菌LF5发酵液抑菌率随着小麦胚芽油浓度的升高明显提升,且该现象具有菌株特异性。通过加热、中和pH、添加蛋白酶及过氧化氢酶发现,乳酸菌协同发酵上清液中的抗真菌活性物质主要由有机酸以及部分具有良好热稳定性的非蛋白类组分组成。此外,采用乳杆菌LF5协同小麦胚芽油发酵制备面包,在第5 d发现肉眼可见霉菌,与空白组(第2~3 d)相较具有显著抑制黑曲霉生长活性的作用,为乳酸菌在发酵米面食品中的应用提供了新的方向。     

麦胚富集γ-氨基丁酸的培养条件优化

采用水浴保温的方法对麦胚中γ-氨基丁酸(GABA)进行富集,研究了培养温度、时间、液料比和培养液pH对麦胚中GABA含量和谷氨酸脱羧酶(GAD)活性的影响,采用响应面法对麦胚富集GABA的培养条件进行了优化。结果表明,在一定范围内培养温度、时间、液料比和培养液pH可有效提高麦胚中GAD活性,促进GABA积累。Box-Behnken实验结果显示,麦胚富集GABA的最优培养条件为培养温度46℃,时间1.5h,液料比6∶1(mL/g),培养液pH4.6。在此培养条件下,麦胚中GABA最大富集量为36.78mg/g,是麦胚原料的5.51倍。方差分析表明,所建的回归模型能够很好的预测麦胚中GABA富集量的变化。     

植物乳杆菌代谢产细菌素的培养基优化

本文对植物乳杆菌代谢产细菌素的培养基进行一系列优化。结果显示,不同培养基对菌株生长和细菌素产量有重要的影响,其中以碳源的影响最为显著。综合考虑,5%糖蜜、0.5%酵母膏、2%胰蛋白胨、0.4%KH2PO4、0.1%MgSO4.7H2O、0.5%CaCO3、0.05%MnSO4和0.3%吐温80是植物乳杆菌生长和代谢产细菌素的最优培养基组合。     

Association of culture of mouse urogenital complexes in media containing rodent sera with the appearance of primordial germ cell-like cells

Primordial germ cells differentiate into germ cells and have the ability to reacquire totipotency. Mouse primordial germ cells are identified by alkaline phosphatase staining of the extraembryonic mesoderm, and they proliferate and migrate to reach the genital ridges. Mouse primordial germ cells have never been maintained in culture exclusively for longer than a week without differentiation or dedifferentiation. Moreover, primordial germ cells have not been proliferated with urogenital complexes in vitro, because gonad culture has never been successful. It was thought that primordial germ cells could proliferate in a culture of urogenital complex under modified medium conditions resembling those in vivo; however, organ culture of mouse gonad has been performed with fetal calf serum or equine serum, and those sera produce conditions different from those in vivo. Therefore, mouse urogenital complexes were cultured in media containing rodent sera. As a result, it was possible to proliferate primordial germ cell-like cells outside gonads, and these cells very closely resembled primordial germ cells. In addition, motile primordial germ cell-like cells could be obtained. The ability to maintain primordial germ cell-like cells in culture by this intra-species culture method is important in the study of gametogenesis. Furthermore, this method is useful as a source of stem cells such as embryonic germ cells.     

Co-culture experiments demonstrate the usefulness of Lactobacillus sakei 10A to prolong the shelf-life of a model cooked ham

This study investigated the usefulness of two selected lactic acid bacteria, Lactobacillus sakei subsp. carnosus (10A) and the lactocin S producing L. sakei 148 (LS5), to extend the shelf-life of cooked meat products. The interaction between these potential protective cultures and the spoilage organisms, Leuconostoc mesenteroides (LM4) and Brochothrix thermosphacta (BT1), were examined in co-culture studies on a model cooked ham product at 7 degrees C under vacuum packaged conditions. Furthermore, the influence of the glucose content of the model cooked ham on the interaction phenomena was investigated. When artificially contaminating the model cooked ham with BT1 at 10(2) cfu/g in combination with 10A at 10(5) cfu/g, the growth of BT1 was significantly slower compared to a simultaneous mono-culture experiment. In a similar experiment with LM4, LM4 reached a level of 10(7) cfu/g +/-14 days later when LM4 grew together with 10A compared to its growth in mono-culture. The lactocin S producing LS5 did not demonstrate an inhibitory action towards LM4 or BT1 and is therefore not useful as protective culture on cooked meat products. The glucose level of the model cooked ham had no influence on the observed antagonistic interactions of 10A towards LM4 or BT1, indicating that the action of the biopreservative 10A in cooked meat products is independent of the substrate glucose.     

ERIC-PCR和Sau-PCR对单增李斯特菌分型稳定性研究

为了研究肠杆菌间重复序列-聚合酶链式反应(ERIC-PCR)和Sau-PCR两种现代分子分型方法对单增李斯特菌(LM)分型的稳定性,取分离自广州市菜市场、超市及厦门某食品加工厂不同食品来源的5株单增李斯特菌进行实验,分别将这5株单增李斯特菌株进行室温放置培养和传代培养,提取室温放置培养24 h、48 h、和72 h及传代培养至第5代、10代、15代和20代的基因组DNA,然后同时进行ERIC-PCR和Sau-PCR分型,观察随着放置时间延长及传代次数增加其指纹图谱的变化。结果显示,5株单增李斯特菌在室温放置培养及传代培养后除部分条带发生缺失外均未出现条带增加现象,整体条带变化不大,两种分型方法的同源性分别在92%和94%以上,表明ERIC-PCR和Sau-PCR两种分型方法在室温放置培养72 h和传代培养20代内对单增李斯特菌分型相对比较稳定,具有流行病学意义。     

Maintenance of adult mouse type A spermatogonia in vitro: influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture

The culture of spermatogonial cells under well-defined conditions would be an important method for elucidating the mechanisms involved in spermatogenesis and in establishing tissue regeneration in vivo. In this study, a serum-free culture system was established, with type A spermatogonia isolated from adult vitamin A-deficient mice. At days 1, 3 and 7 of culture, the viability and proliferation of cells were monitored. The viability of the cells decreased by day 7 to 10% of the cells present. Proliferation occurred mainly during day 1, when 1% of the germ cells was proliferating. Co-labelling for a germ cell marker (heat shock protein-90alpha, Hsp90alpha), and a marker used to detect dividing cells (bromodeoxyuridine, BrdU), showed that this proliferation was restricted to germ cells. In an attempt to improve these parameters, medium containing fetal calf serum (FCS) was used. Viability was not influenced by serum, but proliferation was markedly enhanced. However, after day 7 of incubation with FCS, co-immunolocalization for Hsp90alpha and BrdU showed a preferential proliferation of somatic cells. Comparison of cultures of adult cells with cultures of prepubertal germ cells, commonly used in studies of spermatogenesis, showed that prepubertal germ cells are twice as viable. In addition, a different proliferation profile was observed, with a peak at day 3. Here, a distinct proliferation of somatic cells was also noted. The results from the present study indicate that the origin of isolated germ cells partly determines culture outcome and that cultures of prepubertal germ cells may not be representative for adult spermatogenesis. Moreover, adding FCS to the culture medium invokes the risk of profound and undesirable effects on cell composition, also underlining the need for identification of germ cells during culture.     

The effect of processed meat and meat starter cultures on gastrointestinal colonization and virulence of Listeria monocytogenes in mice

Listeria monocytogenes is a foodborne pathogen of major concern to the food industry in general and the meat industry in particular. The aim of this study was firstly to identify a strain of Listeria that was virulent in SPF BALB/c mice. Secondly, to investigate if a traditional meat starter culture (FloraCarn) and nontraditional meat starter (NTMS) cultures of dairy product and human origin (Lactobacillus and bifidobacteria) inhibit this pathogen in vivo. In addition, the inhibition of Listeria was investigated in vitro. In vitro inhibition was investigated using an agar inhibition assay, where soft agar containing the pathogen was laid over colonies of NTMS cultures, and inhibition expressed as the zones of inhibition developing around the colonies. For assessment of virulence, mice were intragastrically challenged with broth cultures of five strains of Listeria. For assessment of anti-listeria effect in vivo, the Listeria strain proven to be most pathogenic (LM3) was given to mice in salami batter containing no other added cultures (control) or batter inoculated with either (1) FloraCarn, (2) a NTMS culture, or (3) a combination of FloraCarn and a NTMS culture. The batter was given to mice after a 3-day fermentation and faecal levels of pathogen and body weight were monitored. Intragastric challenge with LM3, but no other strains, resulted in a significant weight loss (p<0.05) and up to 10(6) colony forming units (cfu) of LM3 per gram faeces. No weight loss was observed in animals fed with salami batter containing LM3. Consumption of salami batter fermented by a combination of NTMS culture (Lactobacillus acidophilus LAFTI(R) L10) and FloraCarn reduced faecal levels of the pathogen by 2.5 log units compared to the control. Consumption of salami batter fermented with FloraCarn and LAFTI(R) L10 (L10) alone reduced faecal levels by 0.5-1 and 1.5 log units, respectively. Of the NTMS cultures investigated here, L10 displayed the greatest inhibition of LM3 in vitro. These results indicate that the ability of pathogenic Listeria to cause listeriosis is dependent on the nature of the food in which the pathogen is present, and that a traditional meat starter culture (FloraCarn) and some NTMS cultures, particularly L10, inhibit growth of the pathogen during passage through the gastrointestinal tract.     

延长小麦胚贮藏期的研究

小麦胚富含各种保健功能因子,营养价值高,由于贮藏的稳定性差,限制其综合利用。试验利用蒸气微波混合法研究小麦胚的稳定化,结果表明:蒸气微波混合法可以在较短的处理时间内,以较低的能量消耗,达到较好的灭酶效果,起到了稳定小麦胚的作用;试验确定最佳参数为:蒸气处理5min,冷却后560W微波处理5min。添加脱氧剂密封包装于黑暗中贮藏24d后,酸价和酸价变化率分别为5.92和12.8%,均低于对照样品的9.23和99.4%。     

Postmortem degradation of desmin and calpain in breast and leg and thigh muscles from Taiwan black‐feathered country chickens

BACKGROUND: Several studies have reported that the postmortem changes are more rapid in breast muscles (BM) than in leg and thigh muscles (LM) of chickens. However, the reasons for the differences in postmortem proteolysis of BM and LM are still uncertain. The purpose of this study was therefore to compare the postmortem degradation of desmin and calpains in BM and LM from Taiwan black‐feathered country chickens at 5 °C. RESULTS: The pH was lower (P < 0.05) in BM than in LM. Western blot indicated that postmortem desmin degradation was more rapid in BM than in LM. Casein zymograms showed that at‐death µ‐calpain activity was higher in BM than in LM. As postmortem time proceeded, µ‐calpain was activated and autolyzed more extensively in BM than in LM. However, the µ/m‐calpain activity remained stable during postmortem storage in both BM and LM. CONCLUSION: Our results suggest that the more rapid postmortem proteolysis found in BM than in LM at 5 °C similar with the previous study could be mainly explained by both greater amounts and faster activation and autolysis of µ‐calpain in BM. Copyright © 2010 Society of Chemical Industry