COMPARISON OF A REAL-TIME POLYMERASE CHAIN REACTION ASSAY WITH A CULTURE METHOD FOR THE DETECTION OF SALMONELLA IN RETAIL MEAT SAMPLES

A real‐time polymerase chain reaction (PCR) method developed in this study was compared with a conventional International Organization for Standardization culture method for the detection of Salmonella in Irish beef, chicken, pork and turkey. This also provided a snapshot of the incidence of Salmonella in such products. After selective enrichment, all meat samples (n = 100) were: (1) subjected to DNA extraction and real‐time PCR analysis using primers against Salmonella spp. specific gene (16S rRNA) or the invA virulence gene; and (2) plated on differential media and identified as Salmonella using immunological and sub‐typing methods. Retail samples (5/100) were shown to contain Salmonella using the 16S rRNA gene‐based real‐time PCR assay and the standard culture method. However, only two (of five) samples were shown to contain Salmonella using the invA gene‐based real‐time PCR assay. For the sample set examined, the developed 16S rRNA gene‐based real‐time PCR assay demonstrated comparable specificity and sensitivity to the currently used standard culture method but was considerably more rapid.     

A SPECIFIC QUALITATIVE DETECTION METHOD FOR PEANUT (ARACHIS HYPOGAEA) IN FOODS USING POLYMERASE CHAIN REACTION

A qualitative method for detection of peanuts in foods using polymerase chain reaction was developed. A universal primer pair CP 03‐5′/CP 03‐3′ was designed to confirm the validity of the DNAs for PCR. The plant‐specific amplified fragments were detected from 13 kinds of plants using the universal primer pair. In addition, for the specific detection of peanuts with high sensitivity, the primer pair agg 04‐5′/agg 05‐3′ was designed to detect the gene encoding the peanut agglutinin precursor. The primer pair specifically generates a 95‐bp amplified fragment from peanut genomic DNA. Five hundred femto grams of peanut genomic DNA can be detected using the established method. The same qualitative results were obtained from both model processed and nonprocessed food samples containing 0.001, 0.01 and 0.1% of peanut. Moreover, it was shown that the trace amount of peanut in the commercial food products could be qualitatively detected using this method. The reproducibility and applicability of the proposed methods were verified in a six‐laboratory collaborative study.     

QUANTITATIVE DETECTION OF ESCHERICHIA COLI 0157:H7 IN GROUND BEEF BY IMMUNOMAGNETIC SEPARATION AND COMPETITIVE POLYMERASE CHAIN REACTION

ABSTRACT

A protocol based on immunomagnetic separation (IMS) and competitive polymerase chain reaction (C-PCR) was developed for quantitative detection of Escherichia coli 0157:H7 in ground beef. Internal standard sequences were synthesized by PCR with hybrid primers composed in part of original primers for C-PCR. A single concentration of internal standards was used for C-PCR after calibration with known amounts of target DNA. After a 6 h enrichment in Tryptic Soy Broth (TSB), C-PCR enabled quantitative detection of 0.5 to 5.5 CFU per gram of ground beef with Shiga-like toxin 1 and 2 (SLT 1 and 2) primers respectively.     

QUANTITATIVE DETECTION OF ESCHERICHIA COLI 0157:H7 IN GROUND BEEF BY IMMUNOMAGNETIC SEPARATION AND COMPETITIVE POLYMERASE CHAIN REACTION

A protocol based on immunomagnetic separation (IMS) and competitive polymerase chain reaction (C-PCR) was developed for quantitative detection of Escherichia coli 0157:H7 in ground beef. Internal standard sequences were synthesized by PCR with hybrid primers composed in part of original primers for C-PCR. A single concentration of internal standards was used for C-PCR after calibration with known amounts of target DNA. After a 6 h enrichment in Tryptic Soy Broth (TSB), C-PCR enabled quantitative detection of 0.5 to 5.5 CFU per gram of ground beef with Shiga-like toxin 1 and 2 (SLT 1 and 2) primers respectively.     

QUANTITATIVE DETECTION OF ESCHERICHIA COLI O157:H7 IN GROUND BEEF BY IMMUNOMAGNETIC SEPARATION AND POLYMERASE CHAIN REACTION

A quantitative assay for viable Escherichia coli O157:H7 in ground beef based on immunomagnetic separation (IMS) and the polymerase chain reaction (PCR) was developed. Bacterial cells inoculated into ground beef, were captured by immunomagnetic beads (IMB) after a 6 h non-selective enrichment. The percent capture of the target cells was consistent for the inoculation levels of 0.7 to 70 colony-forming-units (CFU)/g. Captured bacteria were lysed with PCR buffer containing 0.2 mg/mL proteinase K at 65°C for 30 min. DNA sequences of Shiga-like toxin 1 and 2 (Stx 1 and 2) were amplified independently. Log-linear relationships were observed between CFU of E. coli O157:H7 inoculated into ground beef and the integrated fluorescent image of the PCR products with Stx 1 and 2 primers after enrichment. The quantitative range was between 0.7 to 70 CFU/g.     

IDENTIFICATION OF BARLEY VARIETIES USING THE POLYMERASE CHAIN REACTION

The polymerase chain reaction (PCR) with oligonucleotide primer sequences from alpha-amylase genes was used to distinguish between morphologically similar malting and feed barley varieties. The varieties “Chebec” (feed) and “Schooner” (malting) could be separated and the varieties “Skiff” (feed) and “Franklin” (malting) were identified by using two sets of primers. Primer BSW3 (5-CAGCTTGGCCTCCGGGCAAGTC-3) and BAS2 (5-CACCTTGCCGTCGATCTC-3) gave a 215 bp product that distinguished “Chebec” from “Schooner” and a 1230 bp fragment that distinguished “Skiff” from “Franklin”. Primer BSW5 (5-GGAGCTGGAATTGATGTTG-3) with primer BAS2 gave markers at 735 bp and 730 bp respectively to allow unique identification in comparisons of these pairs of varieties. DNA extracted from grain could be used for these analyses, but DNA from leaves gave clearer results.     

利用毛细管电泳法测定乳品中乳铁蛋白含量

将样品经样品缓冲液处理,毛细管柱选择为直径50 μm,有效长度400 mm的涂层中性毛细管柱;柱温为40℃;缓冲溶液为柠檬酸缓冲液(pH为3.0±0.2);工作电压为21 kV;检测波长为214nm;进样时间为5s,进行检测.结果乳铁蛋白平均回收率为75%～91%;相对标准偏差(RSD)为1.1%～2.8%;最低检出限位0.003mg/mL.该方法简便,准确,灵敏度高,能快速检测乳品中乳铁蛋白含量.     

抗草甘膦转基因大豆加工品的PCR检测研究

以进口抗草甘膦转基因豆粕、豆粉和中国脱脂大豆为材料,利用设计的特殊引物,建立了PCR技术定性检测抗草甘膦转基因大豆加工品的方法,成功检测出进口抗草甘膦转基因豆粕和豆粉的内源参照基因Lectin、CaMV35S/CTP of EPSPS边界序列和NOS终止子,国产脱脂大豆仅检出内源参照基因Lectin,确定了此PCR技术检测抗草甘膦转基因大豆的灵敏度为0.1%,稳定性良好.     

DETECTION OF HELICOBACTER PYLORI FROM FOOD SOURCES BY A NOVEL MULTIPLEX PCR ASSAY

ABSTRACT

Helicobacter pylori infection is worldwide and it has been recognized as an important cause of gastritis, gastric and duodenal ulcers. However, the modes of transmission of H. pylori infection are unclear. This study was designed to detect H. pylori from raw or ready‐to‐eat foods to provide more evidence for oral‐oral and fecal‐oral transmission patterns. For this purpose, a total of 11 fresh raw chickens from a local grocery and 18 orders of ready‐to‐eat raw tuna meat from a restaurant were collected. H. pylori were detected with our new multiplex polymerase chain reaction (PCR) assay, which can amplify 10 DNA fragments from five gene loci at the same time. H. pylori were positive in 36% (4/11) of the raw chickens and 44% (8/18) of the ready‐to‐eat raw tuna meat tested using the new multiplex PCR assay. Our results demonstrate that food might be a vehicle for H. pylori transmission among humans.

PRACTICAL APPLICATIONS

The prevalence of Helicobacter pylori infection may be as high as 80% in developing countries and up to 40% in developed countries. However, the mode of transmission, the natural history and other aspects of the epidemiology of H. pylori infection are still unclear. There have been no reports of H. pylori being cultured and isolated from foods. In this study we designed a one‐step multiplex polymerase chain reaction assay to amplify 10 DNA fragments at the same time to detect H. pylori, which could overcome the difficulty in identifying H. pylori by using culture or other routine tests. Since this assay is so sensitive, specific and rapid, it has a great potential for identifying H. pylori, which is difficult to be cultured by using current methods, from different food samples.     

猪肉中沙门氏菌的PCR检测

选用合适的引物,利用PCR快速扩增反应检测猪肉中的沙门氏菌.根据沙门氏菌的Fimy基因设计一对引物,对不同血清型的沙门氏菌和非沙门氏菌进行PCR检测,沙门氏菌均能检测出特异条带,而非沙门氏菌无一能检测出特异条带.将沙门氏菌和非沙门氏菌混合培养,对混合培养物提取DNA模板进行检测,结果呈阳性,而不含沙门氏菌的混合培养物则没有特异条带;对模板进行梯度稀释后PCR扩增检测,当DNA含量只有0.045 ng/μL时仍能扩增出条带,将菌液进行梯度稀释后提取DNA模板,沙门氏菌含量在102cfu/mL时能被检测出来.     

德昌水牛乳脂肪酸组成的气相色谱—质谱分析

分析德昌水牛及其杂交后代乳脂肪酸的组成特点.采集德昌水牛及其与尼里拉菲水牛的F1代常乳样品,提取脂肪后进行甲酯化,用气相色谱-质谱联用法测定脂肪酸组成.结果显示,德昌水牛及其F1代乳中的主要脂肪酸为肉豆蔻酸、棕榈酸、油酸、硬脂酸,乳中肉豆蔻酸、棕榈酸比例显著低于F1代,而硬脂酸、油酸比例显著高于F1代.德昌水牛乳脂肪酸组成与牛乳、牦牛乳相比存在一些差异.     

APPLICATION OF THE POLYMERASE CHAIN REACTION TO THE RAPID ANALYSIS OF BREWERY YEAST STRAINS

The rapid discrimination of closely-related Saccharomyces cerevisiae strains can pose a significant problem to breweries, in particular where closely related strains are being used simultaneously to manufacture different products. In this study, two PCR approaches have been examined to assess their usefulness for the discrimination of brewery ale and lager yeast strains. PCR using arbitrary primers (RAPD PCR) was found unsuitable for such an application since the DNA profiles generated from brewery strains were generally found to be identical, due presumably to the close genetic relatedness of these yeasts. In contrast, PCR using δ sequence primers could rapidly differentiate between many ale and lager strains and characteristic profiles for these were generated. This method could also be applied directly to yeasts isolated from brewery worts or from active dried yeast preparations. Results of such analyses were available within the working day.     

IDENTIFICATION OF GOAT MEAT USING HIGHLY SPECIES‐SPECIFIC POLYMERASE CHAIN REACTION

ABSTRACT

A highly species‐specific polymerase chain reaction (PCR) assay was developed for the authentic identification of goat. A product of 436 bp was amplified using newly designed primers against mitochondrial D‐loop region. The possibility of cross‐amplification was ruled out by considering as many as 25 other animal species. Suitability of the developed goat species‐specific PCR assay was confirmed for in raw, cooked (60, 80 and 100C for 30 min) and micro‐oven‐processed meat samples (n = 20 each). A sensitivity of 0.1% was established for detection of adulteration and limit of detection of goat DNA was 0.1 pg. This investigation presents a novel PCR assay with its newly designed primers that could be used for the authentic identification of goat species.

PRACTICAL APPLICATIONS

This work details about a novel diagnostic polymerase chain reaction, which could be used for authentic identification of goat species. This approach could be used for the confirmation of goat tissues in raw, cooked, as well as adulterated samples. The developed technique has also applications in the forensic analysis of wild animal‐related disputes, where this work could solve the problem of goat‐related issues.     

IDENTIFICATION OF MILK FROM GOAT, EWE AND COW IN THEIR MIXTURES BY POLYACRYLAMIDE GEL ELECTROPHORESIS

A rapid identification of milk from goat, ewe and cow was done in the raw milk mixtures by polyacrylamide gel electrophoresis method. Two kinds of milk were mixed in the following proportions: 75:25%, 50:50%, 25:75% (V/V). Raw goat milk was adulterated with increasing proportions of cow milk or ewe milk, and ewe milk was adulterated with cow milk. Ewe milk in goat milk was detected by the electrophoretic resolution of the casein fraction whereas cow milk in goat and in ewe milk was identified by the presence of two fast-moving whey proteins.     

DETECTION OF SALMONELLA TYPHIMURIUM IN OYSTERS BY PCR AND MOLECULAR HYBRIDIZATION

Because shellfish (oysters, clams and mussels) are filter feeders, i.e., able to concentrate pathogens from the surrounding waters within their tissues, they have been widely associated with outbreaks illness. The incidence of salmonellosis caused by the consumption of raw or undercooked shellfish, is a primary concern of public health agencies. Then, in recent years, more rapid and specific methods based on the DNA sequence of salmonella genes have been developed to detect low levels of pathogens in environmental and food samples. In this study, we developed a sensitive method to detect low levels of Salmonella typhimurium in oyster tissues (0.1 cfu/g). This methodology consisted of dissection of the gastrointestinal oyster tract, pre‐enrichment of the samples in nonselective medium, DNA extraction and polymerase chain reaction followed by molecular hybridization using a digoxygenin‐labeled amplicon‐derived probe. These results can benefit the public health agencies and shellfish producers concerning microbiological and quality aspects of the commercial oyster production.     

DETECTION OF ANTIMICROBIAL DRUG RESIDUES IN KENYAN MILK

The improved Dutch tube diffusion test was used to study the occurrence of inhibitory substances in raw bulk milk samples within the Nakuru District in Kenya. Initially the detection limits of the method were verified using milk standards spiked with selected antibiotics. Addition of penicillinase to inhibitor-positive samples was used for preliminary identification of penicillin G-type antibiotics and residue levels were estimated against a standard curve constructed by means of a B. stearothermophilus disc assay. The two-tube test was used to screen 1109 field samples of which 229 (21%) were suspect positive. The identification procedure confirmed 165 samples (14.9%) to contain penicillin G-type residues of which 118 contained levels exceeding the established EU MRL for penicillin G (4 μg/kg). This study indicates that antibiotic residues are prevalent in milk within the Nakuru district of Kenya. It suggests that the improved tube diffusion test in combination with a multiplate system could be useful for qualitative and quantitative identification of antimicrobial drug residues in milk.