Quantification of total and specific gram-negative histamine-producing bacteria species in fish using an MPN real-time PCR method

Quantification of histamine-producing bacteria (HPB) is necessary in order to elucidate the role that HPB play in scombrotoxin (histamine) fish poisoning. We report here the evaluation of a real-time PCR method for the quantification of total and specific Gram-negative HPB species in fish using a most probable number (MPN) format. The species-specific real-time PCR assay was 100% inclusive for independently detecting Morganella morganii, Enterobacter aerogenes, Raoultella planticola/ornithinolytica and Photobacterium damselae and did not cross react with other histamine- or non- histamine-producing bacteria. The efficiency of the reactions in the absence and presence of Spanish mackerel enrichment containing 1 × 10⁶ CFU/ml of background microflora were 93-104 and 92-99%, respectively. The MPN-real-time PCR assay accurately quantified total and specific HPB in spiked mahi-mahi (Coryphaena hippurus) and Spanish mackerel (Scomberomorus maculates) samples. These methods were used to quantify total and specific HPB in naturally contaminated, decomposing mahi-mahi, Spanish mackerel and tuna (Thunnus albacares) samples. The results of this study indicate that MPN-real-time PCR assays can be used to accurately enumerate total and specific HPB in fish samples. These assays can be applied to assess the effectiveness of mitigation strategies and understand the relationship between HPB and histamine production in decomposing fish.     

Quantitative detection of Listeria monocytogenes in raw milk and soft cheeses: Culture-independent versus liquid- and solid-based culture-dependent real time PCR approaches

A culture-independent and two culture-dependent real time (rt-) PCR approaches were developed to quantitatively identify Listeria monocytogenes in raw milk and soft cheeses. The optimised rt-PCR revealed 100% inclusivity and exclusivity. DNA- and cell-based standard curves showed a good linearity of response (R² ≥ 0.987 and R² ≥ 0.998, respectively) for five orders of magnitude (39 × 10⁵ – 3 × 10⁰ genome equivalents and 10⁶–10¹ CFU equivalents, respectively) with about 100% relative accuracy and inter-assay variability ≤0.90%. Up to 1 genome equivalent/and 10 CFU/reaction were quantified in the DNA and cell standard curves, respectively. The rt-PCR was then combined with a liquid- (MPN technique) or a solid- (ALOA and PALCAM) based enumeration. The diagnostic sensitivity of the different approaches was investigated in artificially contaminated raw milk and soft cheeses. The rt-PCR culture-independent approach performed well in raw milk and (with a lower sensitivity) in stracchino cheese-based standard curves. MPN/rt-PCR was the best approach to enumerate low levels of L. monocytogenes in raw milk and stracchino cheese, while the ALOA-based rt-PCR quantification was more effective than the PALCAM-based. These performances were confirmed when 23 real samples of raw milk and soft cheeses by both the rt-PCR approaches were assayed.     

JAK2-V617F突变初步研究

目的用等位基因聚合酶链反应(AS-PCR)方法检测Bcr-abl阴性的骨髓增殖性肿瘤(MPN)、骨髓增生异常综合征(MDS)、骨髓增生异常综合征/骨髓增殖性肿瘤(MDS/MPN)、急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、Bcr-abl阳性的慢性粒细胞白血病(CML)患者和正常健康人对照的外周血白细胞JAK2-V617F基因点突变情况,了解该突变在不同疾病中的表达,并建立敏感特异高效的JAK2-V617F突变的临床检测方法。方法采用AS-PCR方法检测基因组中JAK2-V617F突变,并经基因测序确定。所有MPN患者均行骨髓活检了解其纤维增生度。结果MPN各型中真性红细胞增多症(PV)阳性率为93.8%;原发性血小板增多症(ET)阳性率为60.0%;原发性骨髓纤维化(IMF)阳性率为0;MDS阳性率为4.5%;MDS/MPN阳性率为12.5%;AML、ALL、CML及正常对照均为阴性。MPN各型之间、MPN与其它疾病之间,阳性率差异有显著性(P<0.05)。JAK2-V617F突变阳性的PV及ET患者白细胞数较阴性患者高,差异有显著性(P<0.05)。且阳性患者骨髓纤维组织增生较阴性者明显,差异...     

The Investigation of the Presence of Clostridium Botulinum Spores in Honey in Serbia

The presence of Clostridium botulinum spores in 59 honey samples originating from different regions of the Republic of Serbia was studied. In addition to microbiological methods, after enrichment, centrifugation and membrane filtration, molecular methods (PCR methods) were utilized. The number of spores in PCR positive samples was estimated by the most probable number (MPN) method. PCR confirmed C. botulinum spores in 5 (8.47%) honey samples. MPN of spores varied from 20/kg to 204/kg honey. PCR was more sensitive than cultural methods. Natural honey contamination with C. botulinum spores is low-level and not homogeneous, and therefore, PCR methods require multiple sub-sampling.     

Serial dilution with a confirmation step

A serial dilution test estimates the concentration of a microbe in a broth by inoculating several tubes with portions of the broth. The test may include both presumptive and confirmation steps. For the confirmation step considered here a portion of the contents of each tube indicating a change in the presumptive step is streaked on a plate. Several colonies are selected and examined to determine if the tube contained the target microbe. A statistical model accounts for the possibility of not selecting the target microbe from a tube that contains it and a similar appearing microbe. Simulations show that this model sometimes gives estimates similar to those currently used, but at other times can make large corrections. Also, a commonness measure helps check the validity of the assumptions of this statistical model.     

最大可能数法计数乳品中双歧杆菌数量的研究

目的:建立乳品中双歧杆菌数量快速测定方法。方法:采用莫匹罗星-TPY选择性液体培养基,基于果糖-6-磷酸酮酶酶活测定及最大可能数法确定样品中双歧杆菌数量。结果:该方法所计双歧杆菌数量与平板计数数量在同一数量级。结论:该方法可用于乳品中双歧杆菌的准确计数。     

Platelets Contribution to Thrombin Generation in Philadelphia-Negative Myeloproliferative Neoplasms: The “Circulating Wound” Model

Current cytoreductive and antithrombotic strategies in MPNs are mostly based on cell counts and on patient’s demographic and clinical history. Despite the numerous studies conducted on platelet function and on the role of plasma factors, an accurate and reliable method to dynamically quantify the hypercoagulability states of these conditions is not yet part of clinical practice. Starting from our experience, and after having sifted through the literature, we propose an in-depth narrative report on the contribution of the clonal platelets of MPNs—rich in tissue factor (TF)—in promoting a perpetual procoagulant mechanism. The whole process results in an unbalanced generation of thrombin and is self-maintained by Protease Activated Receptors (PARs). We chose to define this model as a “circulating wound”, as it indisputably links the coagulation, inflammation, and fibrotic progression of the disease, in analogy with what happens in some solid tumours. The platelet contribution to thrombin generation results in triggering a vicious circle supported by the PARs/TGF-beta axis. PAR antagonists could therefore be a good option for target therapy, both to contain the risk of vascular events and to slow the progression of the disease towards end-stage forms. Both the new and old strategies, however, will require tools capable of measuring procoagulant or prohaemorrhagic states in a more extensive and dynamic way to favour a less empirical management of MPNs and their potential clinical complications.     

MPN法测定医疗保健产品生物负载的探讨

本文从医疗保健产品微生物检测的实际需要出发,详细介绍了生物负载MPN法测定的应用进展、使用前提、方法实施及验证。最后总结了MPN法的结果计算公式,并讨论了结果计算中的一些特殊情况,可为生物负载检测提供参考。     

硫酸盐还原菌(SRB)对碳钢管道腐蚀的影响

从现场取回的四种环境介质中提纯SRB，最大可能计数法计数（MPN）结果表明，四种试样中均有SRB，特别是管内流动水中也存在一定数量的SRB，必须采取相应措施灭菌。SRB对碳钢的腐蚀影响与其数量有关。试样表面生成的生物膜较为致密时，对腐蚀有一定的阻碍作用。