Twelve-Hour PCR-Based Method for Detection of Salmonella spp. in Food

A PCR-based method for the detection of Salmonella spp. in food was developed. The method, set up on typical salami from the Italian region of Marche, is sensitive and specific and shows excellent correlation with the conventional method of reference when naturally contaminated foods are analyzed. Moreover, it can be easily performed within a maximum of 12 h from food sampling, thus allowing prompt detection of Salmonella spp. in the food stocks analyzed.     

Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella bacteria in meat

We developed a 12-h Salmonella detection method, based on 8 h of preenrichment, followed by automated DNA extraction and a sensitive real-time PCR. The method was optimized to obtain the highest possible yield of cells and DNA. The growth of different Salmonella strains in various preenrichment media and the effects of adding growth-promoting and selective reagents were explored, taking into account their PCR compatibility. The effects of (i) analyzing larger volumes (1 to 5 ml) from preenriched samples and introducing wash steps prior to DNA extraction, (ii) regulating the amount of paramagnetic particles (increasing it from 60 to 90 microl) in the DNA extraction, (iii) eluting the DNA in reduced volumes (25 or 50 microl rather than 100 microl), and (iv) increasing the PCR template volume (from 5 to 20 microl) were investigated. After 8 h of preenrichment, buffered peptone water yielded the highest number of salmonellae. When analyzing minced meat samples, positive effects of increasing the initial sampling volume from 1 to 5 ml and increasing the amount of paramagnetic particles to 90 microl were observed. However, washing the pellet and eluting the DNA in reduced volumes (25 and 50 microl) had no positive effects and resulted in decreased reproducibility. Increasing the amount of PCR template DNA from 5 to 20 mul improved the threshold cycle value by approximately 2. The improved 12-h PCR method was successfully compared to a reference culture method with 100 minced meat and poultry samples, with a relative accuracy of 99%, a relative sensitivity of 98%, and a relative specificity of 100%.     

PCR法快速检测肉食品污染沙门菌的实验研究

沙门菌属是引起食源性疾病的重要致病菌之一。传统的检测方法费时、费力,研究建立了检测肉食品中沙门菌的PCR检测方法。根据沙门菌侵袭基因invA设计一对引物,对肉食品中沙门菌基因组DNA进行PCR扩增,建立了沙门菌特异、敏感和快速的PCR检测方法,为食源性致病菌的检测提供参照,在食品检测领域中有良好的应用前景。     

Validation of a PCR-Based Method for Detection of Food-Borne Thermotolerant Campylobacters in a Multicenter Collaborative Trial

A PCR-based method for rapid detection of food-borne thermotolerant campylobacters was evaluated through a collaborative trial with 12 laboratories testing spiked carcass rinse samples. The method showed an interlaboratory diagnostic sensitivity of 96.7% and a diagnostic specificity of 100% for chicken samples, while these values were 94.2 and 83.3%, respectively, for pig samples.     

Optimization of Sterilization of Salmonella enteritidis in Meat by Surfactin and Iturin Using a Response Surface Method

In this paper, the sterilization of surfactin and iturin to Salmonella enteritidis was observed, and the optimization of the inactivation of surfactin and iturin to S. enteritidis in meat by a response surface methodology was studied. Results showed that surfactin and iturin had high sterilization to S. enteritidis, whose minimal inhibitory concentration was 6.25 and 12.50 μM respectively. The optimization result indicated that S. enteritidis could be inactivated by five orders of magnitude when the temperature was 4.83°C, the action time was 17.20 h, and the concentration (surfactin/iturin molar ratio 1:2) was 0.69 MIC.     

A Modified PCR-Based Method for Rapid Non-Radioactive Detection of Clinically Important Pathogens

We have devised a sensitive and rapid method for the detection of several bacterial pathogens in clinical specimens using PCR. This method has been named Direct Labeling and Detection Procedure (DLDP) and is based on the direct incorporation of a nonradioactive digoxigenin label (DIG-11-dUTP) into a microbial species-specific gene fragment during amplification. Following amplification, the resulting PCR products are cleansed of nonincorporated DIG-11-dUTP, spotted onto a nylon membrane, fixed by UV-crosslinking and the labeled DNA is visualized by digoxigenin detection reagents. Using cultivated reference bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa) we were able to demonstrate a rapid and sensitive detection of < 20 CFU of bacteria in human secretions (sputum, urine, mucous). The present study suggests that DLDP can be used as a reliable method for indication of bacteria in clinical or environmental specimens with the proviso that the selected corresponding oligonucleotide primers provide amplification of strong species-specific genes.     

Method for Rapid Detection of Cyanogenic Bacteria

An agar plate method is described in which the production of hydrogen cyanide by as many as 50 microbial isolates per plate may be detected. Cyanide produced by the organisms reacts with copper(II) ethylacetoacetate and 4,4′-methylenebis-(N,N-dimethylaniline) in a paper disk suspended above the microbial colonies. Cell growth occurs in depressions in the agar surface, which allows separation of colonies and enhances sensitivity of hydrogen cyanide detection.     

Rapid Quantitative Method for Salmonella Detection in Polluted Waters

A procedure has been developed for the enumeration of salmonellae in polluted waters using several modifications of existing techniques. Confirmation of salmonellae is achieved within 48 hr. This procedure includes selective enrichment in m-Tetrathionate Broth (22 +/- 1 hr), plating on Brilliant Green Sulfa Agar (20 +/- 1 hr), and confirmation by flagellar (H) agglutination of the growth in a mannosecontaining medium (6 +/- 1 hr). An incubation temperature of 41.5 C was used throughout this procedure. Dilution to extinction techniques (most probable number) were employed to enumerate salmonellae. Large sample volumes were concentrated through the use of membrane filters. This technique proved to be rapid and reliable for the enumeration of salmonellae in water, waste water, and waste-water sludges.     

Membrane Filter-Fluorescent-Antibody Method for Detection and Enumeration of Bacteria in Water

A technique which employs nonfluorescing membrane filters and specific fluoresceinisothiocynate-labeled antiserum has been successfully used in the identification and enumeration of known species of Escherichia coli which have been added to natural populations of bacteria found in water. The quantitative results compared favorably with those of standard tests. The use of a dissecting microscope with an external lighting arrangement provided a simple requirement for equipment. This method may be useful in monitoring specific bacterial types from waters which were being monitored for specific pollution.     

蜜蜂残翼病毒TaqMan-MGB探针荧光定量RT-PCR检测方法的建立

蜜蜂残翅病毒(Deform wing virus,DWV)已成为世界上最著名、分布最广、研究最为深入的昆虫病原.虽然DWV以前存在于蜜蜂种群中,但一种新的媒介一外寄生螨Varroa破坏体的到来和全球传播,极大地促进了 DWV的流行病学.为了建立快速、准确且能定量分析的蜜蜂残翅病毒,DWV检测方法,本研究参照GenBank中已登录的DWV高度保守的3C-RdRp基因区域,设计了 1对特异性引物和带有羧基荧光素(Fast Auxiliary Memory,FAM)与淬灭基团(Eclipse)标记的TaqMan探针,并对反应条件进行优化,建立了检测DWV的TaqMan-MGB探针荧光定量RT-PCR检测方法(TaqMan qRT-PCR),同时对该检测方法的特异性、敏感性、重复性进行了试验,并对临床样品进行检测.结果显示:该方法最佳上下游引物和探针浓度分别为12.5μmol/L和10μmol/L,最佳退火温度为59℃;敏感性试验中,对DWV质粒标准品检测下限为2.58×101拷贝/μL;本方法特异性较好,对蜜蜂常见病毒-蜜蜂慢性麻痹病毒(Chronic bee paralysis virus,CBPV)、蜜蜂急性麻痹病毒(Acute paralysis virus,ABPV)、黑蜂王台病毒(Black queen cell virus,BQCV)、囊状幼虫病毒(Sacbrood virus,SBV)、以色列急性麻痹病毒(Israeli acute paralysis virus,IAPV)和中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)无交叉反应;且批内变异系数和批间变异系数均小于2％.利用该方法对49份临床疑似样本进行检测,其中DWV阳性样本为35份,检出率高于常规RT-PCR方法,且病毒载量大于1×107拷贝/只样本数共24份,表明辽宁和河北部分地区感染DWV蜂群中病毒载量较高,流行情况比较严重.因此,本研究建立的TaqMan探针荧光定量RT-PCR方法具有灵敏度高、特异性强、重复性好等特点,能够用于DWV病原监测、流行病学调查和病毒定量分析等相关研究.     

Optimization of Multiple Pathogen Detection Using the TaqMan Array Card: Application for a Population-Based Study of Neonatal Infection

Identification of etiology remains a significant challenge in the diagnosis of infectious diseases, particularly in resource-poor settings. Viral, bacterial, and fungal pathogens, as well as parasites, play a role for many syndromes, and optimizing a single diagnostic system to detect a range of pathogens is challenging. The TaqMan Array Card (TAC) is a multiple-pathogen detection method that has previously been identified as a valuable technique for determining etiology of infections and holds promise for expanded use in clinical microbiology laboratories and surveillance studies. We selected TAC for use in the Aetiology of Neonatal Infection in South Asia (ANISA) study for identifying etiologies of severe disease in neonates in Bangladesh, India, and Pakistan. Here we report optimization of TAC to improve pathogen detection and overcome technical challenges associated with use of this technology in a large-scale surveillance study. Specifically, we increased the number of assay replicates, implemented a more robust RT-qPCR enzyme formulation, and adopted a more efficient method for extraction of total nucleic acid from blood specimens. We also report the development and analytical validation of ten new assays for use in the ANISA study. Based on these data, we revised the study-specific TACs for detection of 22 pathogens in NP/OP swabs and 12 pathogens in blood specimens as well as two control reactions (internal positive control and human nucleic acid control) for each specimen type. The cumulative improvements realized through these optimization studies will benefit ANISA and perhaps other studies utilizing multiple-pathogen detection approaches. These lessons may also contribute to the expansion of TAC technology to the clinical setting.     

A Method for the Detection of Starch Hydrolysis by Bacteria

S ummary . A starch agar medium for the detection of starch hydrolysis is described. The development of a cloudy zone round the colony indicates starch hydrolysis without the use of iodine or 95% (v/v) ethanol.     

Method for Radiorespirometric Detection of Bacteria in Pure Culture and in Blood

Methods are described for the detection of low numbers of bacteria by monitoring (14)CO(2) evolved from (14)C-labeled substrates. Cell suspensions are filtered with membrane filters, and the filter is then moistened with 0.1 ml of labeled medium in a small, closed apparatus. Evolved (14)CO(2) is collected with Ba(OH)(2)-moistened filter pads and assayed with conventional radioactivity counting equipment. The kinetics of (14)CO(2) evolution are shown for several species of bacteria. Fewer than 100 colony-forming units of most species tested were detected in 2 h or less. Bacteria were inoculated into blood and the mixture was treated to lyse the blood cells. The suspension ws filtered and the filter was placed in a small volume of labeled medium. The evolved (14)CO(2) was trapped and counted. A key development in the methodology was finding that an aqueous solution of Rhyozyme and Triton X-100 produced lysis of blood but was not detrimental to bacteria.     

Quantification of Salmonella spp. in composted biosolids using a TaqMan qPCR assay

Composting is increasingly used to transform biosolids, obtained following wastewater treatment, into a more stable organic product that can be released in the environment. The process must however be closely monitored to assure that the end product meets the regulations set by environmental agencies with regards to the amount of pathogenic microorganisms present. In this study, a TaqMan qPCR approach targeting the invA gene was developed to monitor the presence of Salmonella spp. in composted biosolids. A validation step was first performed to evaluate the effect of compost age on the quantification of various concentrations of seeded Salmonella typhimurium. Secondly, qPCR was used to investigate the effect of composting time, varying from 1 month to 24 months, on the presence of Salmonella spp. naturally present in biosolids samples. Culture media were used in parallel to corroborate the results obtained by qPCR. The detection limit of the invA gene obtained experimentally from composts seeded with S. typhimurium was 5.8 copies or the equivalent of 5.8 CFU per qPCR reaction. Although the results indicated that compost age had a marginal effect on the detection of seeded S. typhimurium, the TaqMan qPCR approach was efficient at detecting and quantifying the amount of Salmonella spp. present in naturally contaminated composted biosolids of different ages. Results showed that there was a significant decrease in the amount of Salmonella DNA present in composted biosolids over time, which was also corroborated by the CFU counts obtained on the BSA culture medium. However, qPCR was more specific, robust and rapid to execute than performing counts on culture media. qPCR shows promise for routine examination of composted biosolids to ascertain that pathogenic microorganisms, including Salmonella spp., are decreased below acceptable limits before their application in the environment.     

Improved Method for Detection of Methanotrophic Bacteria in Forest Soils by PCR

A primer set was designed for the specific detection of methanotrophic bacteria in forest soils by PCR. The primer sequences were derived from highly conservative regions of the pmoA gene, encoding the α-subunit of the particulate methane monooxygenase present in all methanotrophs. In control experiments with genomic DNA from a collection of different type I, II, and X methanotrophs, it could be demonstrated that the new primers were specific for members of the genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, and Methylococcus. To test the suitability of the new primers for the detection of particulate methane monooxygenase (pMMO) containing methanotrophs in environmental samples we used DNA extracts from an acid spruce forest soil. For simple and rapid purification of the DNA extracts, the samples were separated by electrophoresis on a low-melting-point agarose gel. This allowed us to efficiently separate the DNA from coextracted humic acids. The DNA from the melted agarose gel was ready for use in PCR reactions. In PCR reactions with DNA from the Ah soil layer, products of the correct size were amplified by PCR by use of the new primers. By sequencing of cloned PCR products, it could be confirmed that the PCR products represented partial sequences with strong similarity to the pmoA gene. The sequence was most related to the pmoA sequence of a type II methanotroph strain isolated from the Ah layer of the investigated soils. Received: 1 September 2000 / Accepted: 2 October 2000     

Improved Template Preparation for PCR-Based Assays for Detection of Food-Borne Bacterial Pathogens

Shigella flexneri, Salmonella enterica serotype Typhimurium, and Listeria monocytogenes were applied to FTA filters, and the filters were used directly as templates to demonstrate their sensitivity and applicability in PCR-based detection assays. With pure cultures, the sensitivities of detection by FTA filter-based PCR were 30 to 50 and 200 CFU for the gram-negative enterics and Listeria, respectively. Different numbers of S. flexneri cells were used in controlled contamination experiments with several different foods (produce, beef, and apple cider). Aliquots from concentrated food washes subsequently spotted onto FTA filters and assayed by PCR gave consistently positive results and detection limits similar to those observed with pure-culture dilutions. This universal method for PCR template preparation from bacterial cells is rapid and highly sensitive and reduces interference from food-associated inhibitors of PCR. In addition, its broad applicability eliminates the need for multiple methods for analysis of food matrices.     

从冻煮青柳蛤肉中检出阿贡纳沙门菌及定量分析

为了掌握丹东口岸进出口冷冻水产品中沙门菌的污染情况和污染程度,采用GB/T 4789.4-2003前增菌和mini-VADIS法快速筛选,对阳性的样品用选择性琼脂平板划线分离并挑取可疑菌落,进行BBL.crysta自动细菌分析鉴定.采用此法从一批冻煮青柳蛤肉中检出了阿贡纳沙门菌,经 MPN法计数结果为2.3/g.该血清型是引起畜禽类和人类沙门菌病传染的重要血清型,应加强对进出口冷冻水产品的卫生监测.     

Development of a Direct In Situ PCR Method for Detection of Specific Bacteria in Natural Environments

We applied HNPP (2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate) to direct in situ PCR for the routine detection of specific bacterial cells at the single-cell level. PCR was performed on glass slides with digoxigenin-labeled dUTP. The digoxigenin-labeled PCR products were detected with alkaline phosphatase-labeled antidigoxigenin antibody and HNPP which was combined with Fast Red TR. A bright red fluorescent signal was produced from conversion to HNP (dephosphorylated form) by alkaline phosphatase. We used the ECOL DNA primer set for amplification of ribosomal DNA of Escherichia coli to identify cells specifically at the single-cell level in a bacterial mixture. High-contrast images were obtained under an epifluorescence microscope with in situ PCR. By image analysis, E. coli cells in polluted river water also were detected.     

Development of a Temperature-Switch PCR-Based SNP Typing Method for Mycobacterium ulcerans

Mycobacterium ulcerans (M. ulcerans), the causative agent of the devastating skin disease Buruli ulcer (BU), is characterized by an extremely low level of genetic diversity. Recently, we have reported the first discrimination of closely related M. ulcerans variants in the BU endemic Densu River Valley of Ghana. In the study real-time PCR-based single nucleotide polymorphism (SNP) typing at 89 predefined loci revealed the presence of ten M. ulcerans haplotypes circulating in the BU endemic region. Here we describe the development of temperature-switch PCR (TSP) assays that allow distinguishing these haplotypes by conventional agarose gel-based analysis of the PCR products. After validation of the accuracy of typing results, the TSP assays were successfully established in a reference laboratory in Ghana. Development of the cost-effective and rapid TSP-based genetic fingerprinting method will thus allow investigating the spread of M. ulcerans clones by regular genetic monitoring in BU endemic countries.