An improved 16S rRNA based PCR method for the specific detection of Salmonella enterica

A molecular method for the detection of Salmonella enterica strains based on 16S rRNA sequence analysis was developed by a modification of the previously described PCR primer 16SFI [J. Appl. Bacteriol. 80 (1996) 659], which was combined with a newly developed primer annealing at the position 66-82. Only approximately two thirds of now determined Salmonella 16S rRNA sequences contained a region identical to the 16SFI primer sequence and the reverse primer 16SIII was also not specific. Combined, these two primers have been claimed to allow the specific detection of all Salmonella; however, in this study, they did not recognize S. bongori and 3 out of 78 tested S. enterica strains. They also identified some of the tested Enterobacter cloacae strains as Salmonella. On the contrary, the new primer pair, MINf and MINr, made it possible to recognize correctly all of the 78 tested S. enterica strains, representing 31 different Salmonella serovars. None of the 23 non-Salmonella strains from the related gamma-proteobacterial genera was incorrectly recognized as belonging to S. enterica.     

The sequence heterogenicities among 16S rRNA genes of Salmonella serovars and the effects on the specificity of the primers designed

Previously, we have reported a 16S rDNA targeted polymerase chain reaction (PCR) method for the specific detection of Salmonella serovars [J. Appl. Bacteriol. 80 (1996) 659]. The target sites of its primers, i.e. 16SFI and 16SIII, according to the data in GenBank, were found mismatched to the corresponding sequences of some Salmonella serovars, such as those of S. Houten, S. Chingola, S. Bareilly, and S. Weltevreden. Accordingly, a PCR method using a nonspecific primer MINf combined with a primer modified from our 16SFI primer, i.e. the primer MINr, was developed and displayed better detection specificity [Int. J. Food Microbiol. 80 (2003) 67]. In this study, we show the sequence heterogenicity at the primer 16SFI targeting sites for some Salmonella serovars. Thus, the sequence used for designing of PCR primers might be just one of the several possible sequences. Such a situation may lead to the misjudgment on evaluation of the specificity of the primers if this was only based on the data in GenBank. Strains of the above described Salmonella serovars with target sequences from GenBank mismatched to the primer 16SF1 were reidentified and their PCR results were confirmed. Meanwhile, their 16SFI/16SIII primer annealing sites were sequenced and the sequences obtained were found completely and highly homologous to those of 16SFI and complementary to those of 16SIII primer, respectively.     

Sequencing of an internal transcribed spacer region of 16S-23S rRNA gene and designing of PCR primers for the detection of Salmonella spp. in food

DNA sequences of an internal transcribed spacer (ITS) region for 40 Salmonella serovars were determined and compared with ITS sequences of Salmonella spp., and non-Salmonella spp. already available on the GenBank database. From such comparison, two Salmonella-specific ITS based PCR primers, ITSF and ITSR, were designed. When Salmonella strains with various serotypes were PCR assayed with primers ITSF/ITSR, all generated PCR products with molecular weight bands equal to 312 bp. On the other hand, 48 non-Salmonella isolates, including strains of Enterobacteriaceae and other food pathogens generated negative results. Detection limits of this PCR method was 1-9 CFU per assay. These PCR primers were used for the detection of Salmonella cells in artificially contaminated foods, including chicken meat and whole milk. The detection limit was 1-9 x 10(3) CFU per assay. With an 8-h enrichment step performed prior to the PCR assay, however, the detection limit became 1-9 CFU per gram of the food sample.     

Development and use of polymerase chain reaction for the specific detection of Salmonella Typhimurium in stool and food samples.

Salmonella Typhimurium is one of the most important Salmonella serovars that may cause foodborne disease and human salmonellosis infection. Detection of this organism in the clinical samples of persons with gastroenteritis and the food samples associated with such persons may allow us to trace the cause of disease. Because malic acid dehydrogenase, an enzyme of the citric acid cycle, is common to organisms, the gene (mdh) coding for this enzyme was selected for the design of Salmonella Typhimurium-specific polymerase chain reaction (PCR) primers. By comparison of the mdh gene sequences of Salmonella Typhimurium and other Salmonella serotypes and of some isolates of other genera, two oligonucleotides were designed and used as PCR primers for the specific detection of Salmonella Typhimurium. The molecular weight of the PCR product was 261 bp as expected. Salmonella serovars other than Salmonella Typhimurium and isolates of other genera in the Enterobacteriaceae that is closely related to Salmonella did not generate any false-positive results. When this primer pair was used for the detection of Salmonella Typhimurium cells artificially inoculated into human stool specimens and food samples, such as milk and raw chicken meat, levels as low as 10(0) CFU per 0.1 g of stool specimen or per ml of milk or food homogenate could be detected if an 8- to 12-h preculture step using combined lactose-tetrathionate broth was performed prior to the PCR.     

Primers specific for the fimbrial major subunit gene stdA can be used to detect Salmonella enterica serovars

The feasibility of using two primers internal to the stdA gene (which encodes the fimbrial major subunit of the std fimbrial gene cluster in Salmonella enterica serovar Typhi) to detect Salmonella by PCR was explored. The 518-bp stdA specific sequence was conserved among 268 strains from 45 serovars of S. enterica. One Salmonella bongori CCUG 30042 strain and 34 non-Salmonella strains did not possess this sequence. A sensitivity test revealed that the stdA-specific primer set detected 3.4 x 10(-1) pg of genomic DNA and 3.0 x 10(5) CFU/ml with serial dilutions of Salmonella Typhimurium cells. In vitro testing for specificity using pig carcass sponge samples contaminated with Salmonella Typhimurium also was performed. An initial Salmonella Typhimurium inoculum of 4.4 x 10(1) CFU/ml in pig carcass exudates reached the stdA primer detection level after preenrichment in buffered peptone water at 37 degrees C for 18 h in the presence of indigenous non-Salmonella flora at 4.0 X 10(7) CFU/ml, but the detection level decreased to 4.4 x 10(0) CFU/ml after selective enrichment in Rappaport-Vassiliadis R10 broth for 18 h at 42 degrees C. The PCR method with primers specific for stdA is a quick and sensitive tool for detecting S. enterica, which is an important cause of foodborne disease.     

Development of PCR primers for the detection of Salmonella enterica serovar Choleraesuis based on the fliC gene

Salmonella enterica serovar Choleraesuis may cause swine salmonellosis and human infection. Because the conventional method for detection of this Salmonella serovar may take 3 to 5 days, a PCR method for detection was evaluated. By comparing the sequence of the phase 1 flagellin (fliC) gene of Salmonella Choleraesuis with that of other Salmonella serovars and of other bacteria species available in GenBank, two PCR primers (flinC-F and flinC-R) were designed. Using these primers, all 97 Salmonella Choleraesuis strains assayed generated the expected PCR product, with a molecular mass of 963 bp. Except for S. enterica Paratyphi C, Salmonella isolates other than Salmonella Choleraesuis and non-Salmonella isolates, including strains of Enterobacteriaceae, all generated negative PCR results. Salmonella Paratyphi C could be differentiated from Salmonella Choleraesuis through the use of primers designed from the viaB gene. When Salmonella Choleraesuis isolates from swine stool, pork, liver, feed, and human whole blood samples were assayed with a preenrichment step, as low as 1 CFU/g or ml of the original sample could be detected.     

Evaluation of TaqMan PCR assay for detecting Salmonella in raw meat and shrimp.

We evaluated the TaqMan Salmonella amplification/detection kit from PE Applied Biosystems, which uses a polymerase chain reaction (PCR) assay for rapid detection of Salmonella in food samples. This system uses the 5' nuclease activity of Taq DNA polymerase, which digests an internal fluorogenic probe to monitor the amplification of the target gene. The system's sensitivity and specificity were evaluated using 42 serotypes of 68 Salmonella strains isolated from fecal samples from patients in Tokyo, Japan, and 39 non-Salmonella strains in 22 genera. There were no false-negative or false-positive results. This PCR assay can detect 3 CFU per PCR tube of Salmonella in pure culture (120 CFU/ml of TSB culture). PCR signals were attenuated with artificially contaminated shrimp, but a similar detection limit was obtained. TaqMan's performance was tested with 100 meat and chicken samples purchased from stores in Tokyo. Overall, two of the DNA extraction protocols (the Chelex and EnviroAmp methods) worked equally well, with some exceptions. Of the 100 samples analyzed, 10 were positive for Salmonella with both conventional culture methods and the kit and 89 were negative with both. One sample was negative by the culture method but positive by the kit assay. These results indicate that TaqMan is a reliable and rapid method for Salmonella analysis in the food industry. With this system, food samples can be analyzed for Salmonella in less than 20 h.     

聚合酶链反应检测食品中的沙门氏菌

参考沙门氏菌侵袭性基因invA的序列,设计合成一对引物扩增其中一段序列,建立了特异性检测沙门氏菌的PCR方法。引物两端分别加了Bam HI和EcoR I切点,扩增片段大小为300bp。对收集的50个血清型123株沙门氏菌及7种23株非沙门氏菌进行PCR检测,结果仅沙门氏菌有300bp的扩增产物,显示了很强的特异性,为下一步克隆而设计的两个酶切位点对引物的特异性没有影响。经琼脂糖凝胶电泳,PCR的检出极限是10pg染色体DNA和10~2 cfu的细菌。将扩增产物经slot—blot与辣根过氧化物酶(HRP)直接标记的invA基因探针杂交,经增强型化学发光反应(ECL)及化学发光自显影(CPD)检测,可提高检测的敏感性一个数量级,同时增加了特异性。为推广应用,本文试验了8种模拟食品样品对PCR反应的影响,结果除奶酪外,其余都得到较好扩增。对120份污水及食品样品进行检测,该检测系统检出70份阳性结果,检出率高于常规分离。初步应用显示,PCR检测方法敏感、特异、简便、快速,适合于食品卫生检验和临床标本检验的现场应用。     

沙门氏菌商品化核酸检测试剂盒评价

目的结合不同标准和参数,对沙门氏菌商品化检测试剂盒进行评价。方法用52个血清型的64株沙门氏菌和20个种属的30株非沙门氏菌,对沙门氏菌商品化核酸检测系统进行包容性和排他性评价;通过与我国国标方法的平行检测,使用20个食品样本对沙门氏菌商品化核酸检测系统进行方法一致性评价。结果商品化检测系统对沙门氏菌的平均检出限为1.51×103 CFU/m L,灵敏度为100%;对30株非沙门氏菌进行检测,商品化核酸检测系统检测结果均为阴性,特异性为100%;对生肉和熟肉制品食品样品进行沙门氏菌检测,沙门氏菌商品化核酸检测系统与国标方法的一致性均在95%以上,均无显著性差异(P0.05),准确度为100%。结论沙门氏菌商品化检测系统以其快速、简便、准确、不需要大型仪器,值得在食品微生物检测行业推广。     

实时荧光PCR法检测鼠伤寒沙门氏菌

目的 建立实时荧光PCR法检测鼠伤寒沙门氏菌的方法。方法 基于鼠伤寒沙门氏菌II型限制酶基因, 设计引物及Taqman探针, 利用实时荧光PCR进行特异性、灵敏性及模拟样品的检测实验。结果 特异性探针可从25种血清型沙门氏菌(共49株)及11株阴性对照菌株中检测出全部的11株鼠伤寒沙门氏菌。以鼠伤寒沙门氏菌梯度稀释菌液DNA为模板进行实时荧光PCR实验, 菌株模板浓度与Ct值呈良好线性关系, 线性系数(R2)为0.998, 扩增效率90%, 最低检测浓度300 cfu/mL。对已接种鼠伤寒沙门氏菌的4种模拟样品同时进行实时荧光PCR检测和传统方法鉴定, 两者结果一致。结论 此方法特异、灵敏、准确, 适于食品中鼠伤寒沙门氏菌的检测。     

Evaluation of three different molecular markers for the detection of Staphylococcus aureus by polymerase chain reaction

The aim of this study was to target three genes of Staphylococcus aureus-fmhA (coding for a factor of unknown function), catalase and femA (coding for a factor essential for methicillin resistance) to establish and validate a PCR assay for the detection of this pathogen. Two pairs of primers were designed for fmhA and one pair each for catalase and femA genes. The PCR assays were standardized and found to give specific amplicons under similar reaction parameters. Target specificity of the primers was confirmed by DNA sequencing of the amplicons. While the initial inclusivity and exclusivity test reactions were in agreement in case of three of the primer pairs, one pair based on fmhA gene produced a non-specific product with a template DNA used in exclusivity test reactions. Forty-five strains of S. aureus were subjected to these PCR assays for their evaluation. Three among the four pairs of primers, one against each gene detected all the 45 strains precisely whereas one of the PCR assays using primers targeting the fmhA gene did not generate the specific amplicon with several of the strains. Seven unidentified strains of Gram-positive cocci subjected to these PCR assays produced negative results for each culture. Six of the strains were identified as Staphylococcus haemolyticus and one strain as Staphylococcus arlettae by 16S ribosomal gene analyses. All the three assay systems showed a detection limit of 100 cells per 20mul reaction assay. For validation of these assay systems, 80 coded samples of 11% skimmed milk spiked with different pathogens were received from NICED (National Institute of Cholera and Enteric Diseases), Kolkata and subjected to these PCR assays. All the three assays could detect S. aureus correctly in two of the samples. Amongst 150 raw milk samples, 36 (24%) were found positive for S. aureus. We conclude that fmhA, catalase and femA genes are conserved in S. aureus and, therefore, could be used as specific targets for its detection and identification by PCR. The protocols developed herein could be used for rapid and specific detection of this pathogen in food, clinical and environmental samples, especially milk.     

双正交优化多重PCR检测食源性致病菌的研究

为了建立快速检测食源性致病菌的多重PCR检测方法,根据沙门氏菌fimY基因、单增李斯特氏菌hlyA基因和小肠结肠炎耶尔森氏菌ail基因设计3对特异性引物,利用双正交法分别对多重PCR反应体系中的3对引物比进行L9(33)正交优化和Taq酶、dNTPs、镁离子、混合引物的添加量进行L16(44)正交优化,并对Buffer的反应浓度进行优化。结果扩增出了3条特异性目的条带,建立的多重PCR方法具有很好的特异性。人工污染食品,9h富集培养增菌后的检出限可达100cfu/g。因此,该检测方法具有较强的实际应用价值,为检测多种食源性致病菌提供了有效的方法参考。     

两种选择性培养基HE、CAS检测沙门菌效果的比较

目的 比较两种沙门菌选择性培养基HE和CAS在食物样本检测中的敏感性、准确性和时效性.方法 观察典型沙门菌和非沙门菌在两种选择培养基上的生长状况和菌落形态.通过人工染菌实验,比较两种选择性培养基分离沙门菌的检测效果.采集16份市售整鸡样本,进一步比较两种培养基沙门菌的阳性检出率.结果 沙门菌在HE平板上为绿色菌落,大多有黑心;在CAS平板上为紫色菌落.含菌量为10 CFU/25 g鸡肉样本,经SC增菌液增菌培养8h,可用CAS检出沙门菌;而采用HE则需增菌18 h才可检出.16份整鸡样本,增菌8,12和18 h,采用CAS的阳性检出率分别为43.75%、31.25%和37.5%,相应时间下采用HE的阳性检出率分别为18.75%,25%和18.75%,8份新鲜整鸡样本,检出4份阳性;8份冷冻整鸡样本,检出3份阳性.结论 CAS选择性平板可抑制大多杂菌生长,沙门菌在其上菌落形态较易鉴别.CAS分离沙门菌的检出时限和检测灵敏度都明显优于HE,建议在食物样本检测沙门菌时应首选CAS.     

甲型副伤寒沙门菌特异性基因筛选及PCR检测体系建立

以甲型副伤寒沙门菌为检测目标,通过比较基因组和聚合酶链式反应（polymerase chain reaction,PCR）验证方法筛选到4 个该血清型的特异性基因,其中以gene_3105作为该血清型的检测靶点设计引物PA23;并结合沙门菌属特异性引物139-141,建立一种甲型副伤寒沙门菌的PCR检测方法。优化PCR反应体系,并对该检测体系的特异性、灵敏度、抗干扰能力及人工污染样品检出限等方面进行评价。结果表明,当样品中含有甲型副伤寒沙门菌时,该体系能扩增出2?条特异性条带,含有其他血清型的沙门菌仅能扩增出284?bp条带,不含沙门菌无扩增条带产生。灵敏度评价表明,基因组DNA和纯菌菌落检出限分别为32.4?pg/μL和4.3×103?CFU/mL;抗干扰能力实验显示,当鸡肉背景菌群和猪肉背景菌群浓度在106?CFU/mL和4.87×107?CFU/mL时,检出限为6.43×104?CFU/mL。当无菌的鸡肉和猪肉样品中添加N?CFU/25?g甲型副伤寒沙门菌时,经10?h增菌,检测结果为阳性（0＜N＜10）。实验建立甲型副伤寒沙门菌PCR检测方法具有较好的特异性和灵敏度,有很好的应用价值,可在食品安全领域广泛应用。     

PCR amplification of the Salmonella typhimurium fimY gene sequence to detect the Salmonella species

This study evaluated the suitability of fimY gene amplification by PCR as an effective means of detecting Salmonella species. Although fimY gene of Salmonella typhimurium is involved in regulating type 1 fimbrial expression, the amino acid sequence of FimY shares very little homology with other known prokaryotic proteins in the GenBank database. Therefore, fimY is a promising target gene to detect the presence of Salmonella species. Herein, two primers internal to the fimY gene of S. typhimurium are used to investigate the distribution of the fimY homologous sequence among 45 Salmonella serovars and 20 non-Salmonella species by using PCR. Experimental results indicated that only Salmonella species possessed the fimY homologous sequence, subsequently generating the specific 526-bp DNA fragments. The sensitivity of the fmY-specific primer set was demonstrated on a Salmonella-free swab sample from a pork carcass surface, which was then artificially contaminated with different concentrations of S. typhimurium. A combining of pre-enrichment step in buffered peptone water and PCR amplification of fimY allowed the detection of S. typhimurium at the concentration of 3.4 x 10(0) CFU/ml from the swab sample. With an additional enrichment step in Rappaport-Vassiliadis (RV) broth, this procedure can also detect pork carcass surface naturally contaminated with Salmonella species in a slaughterhouse. Results in this study demonstrate that fimY is unique to Salmonella species and is an appropriate PCR target for detecting these microorganisms.     

Validation of a PCR-based method for the detection of various rendered materials in feedstuffs using a forensic DNA extraction kit

A method trial was initiated to validate the use of a commercial DNA forensic kit to extract DNA from animal feed as part of a PCR-based method. Four different PCR primer pairs (one bovine pair, one porcine pair, one ovine primer pair, and one multispecies pair) were also evaluated. Each laboratory was required to analyze a total of 120 dairy feed samples either not fortified (control, true negative) or fortified with bovine meat and bone meal, porcine meat and bone meal (PMBM), or lamb meal. Feeds were fortified with the animal meals at a concentration of 0.1% (wt/wt). Ten laboratories participated in this trial, and each laboratory was required to evaluate two different primer pairs, i.e., each PCR primer pair was evaluated by five different laboratories. The method was considered to be validated for a given animal source when three or more laboratories achieved at least 97% accuracy (29 correct of 30 samples for 96.7% accuracy, rounded up to 97%) in detecting the fortified samples for that source. Using this criterion, the method was validated for the bovine primer because three laboratories met the criterion, with an average accuracy of 98.9%. The average false-positive rate was 3.0% in these laboratories. A fourth laboratory was 80% accurate in identifying the samples fortified with bovine meat and bone meal. A fifth laboratory was not able to consistently extract the DNA from the feed samples and did not achieve the criterion for accuracy for either the bovine or multispecies PCR primers. For the porcine primers, the method was validated, with four laboratories meeting the criterion for accuracy with an average accuracy of 99.2%. The fifth laboratory had a 93.3% accuracy outcome for the porcine primer. Collectively, these five laboratories had a 1.3% false-positive rate for the porcine primer. No laboratory was able to meet the criterion for accuracy with the ovine primers, most likely because of problems with the synthesis of the primer pair; none of the positive control DNA samples could be detected with the ovine primers. The multispecies primer pair was validated in three laboratories for use with bovine meat and bone meal and lamb meal but not with PMBM. The three laboratories had an average accuracy of 98.9% for bovine meat and bone meal, 97.8% for lamb meal, and 63.3% for PMBM. When examined on an individual laboratory basis, one of these four laboratories could not identify a single feed sample containing PMBM by using the multispecies primer, whereas the other laboratory identified only one PMBM-fortified sample, suggesting that the limit of detection for PMBM with this primer pair is around 0.1% (wt/wt). The results of this study demonstrated that the DNA forensic kit can be used to extract DNA from animal feed, which can then be used for PCR analysis to detect animal-derived protein present in the feed sample.     

Species-specific identification of penicillium linked to patulin contamination

Certain species of Penicillium have been reported to produce the mycotoxin patulin, and research was undertaken to identify these with the use of oligonucleotide primer pairs. Species examined were found in food, plants, and soil and were reported to produce patulin. Penicillium expansum is the most commonly detected species linked to the presence of patulin in apple juice. At least 10 different enzymes are involved in the patulin biosynthetic pathway, including the isoepoxydon dehydrogenase (idh) gene. Based on nucleotide sequences previously determined for the idh gene in Penicillium species, PCR primers were designed for the species-specific detection of patulin-producing species. The 5' primers were based on differences in the second intron of the idh gene. To ensure that the primer pairs produced a PCR product restricted to the species for which it was designed, and not to unrelated species, all of the primer pairs were tested against all of the Penicillium species. With one exception, it was possible to detect a reaction only with the organism of interest. The primer pair for Penicillium griseofulvum also amplified DNA from Penicillium dipodomyicola, a closely related species; however, it was possible to distinguish between these two species by doing a second amplification, with a different primer pair specific only for P. dipodomyicola. Consequently, with different primer sets, it was possible to identify individual patulin-producing species of Penicillium.     

Identification of Salmonella enterica serovar Typhimurium using specific PCR primers obtained by comparative genomics in Salmonella serovars

Salmonella enterica serovar Typhimurium is a major foodborne pathogen throughout the world. Until now, the specific target genes for the detection and identification of serovar Typhimurium have not been developed. To determine the specific probes for serovar Typhimurium, the genes of serovar Typhimurium LT2 that were expected to be unique were selected with the BLAST (Basic Local Alignment Search Tool) program within GenBank. The selected genes were compared with 11 genomic sequences of various Salmonella serovars by BLAST. Of these selected genes, 10 were expected to be specific to serovar Typhimurium and were not related to virulence factor genes of Salmonella pathogenicity island or to genes of the O and H antigens of Salmonella. Primers for the 10 selected genes were constructed, and PCRs were evaluated with various genomic DNAs of Salmonella and non-Salmonella strains for the specific identification of Salmonella serovar Typhimurium. Among all the primer sets for the 10 genes, STM4497 showed the highest degree of specificity to serovar Typhimurium. In this study, a specific primer set for Salmonella serovar Typhimurium was developed on the basis of the comparison of genomic sequences between Salmonella serovars and was validated with PCR. This method of comparative genomics to select target genes or sequences can be applied to the specific detection of microorganisms.     

Evaluation of a 24-hour bioluminescent enzyme immunoassay for the rapid detection of Salmonella in chicken carcass rinses

A bioluminescent enzyme immunoassay (BEIA), using Salmonella-specific monoclonal antibody M183 for capture and biotinylated monoclonal antibody M183 for detection, was developed with InteLite AB streptavidin-biotinylated firefly luciferase complex as a reporter. Salmonella cultures were preenriched in buffered peptone water with shaking for 6 h at 37 degrees C and then selectively enriched in Muller-Kauffmann tetrathionate (MKTT) broth and modified semisolid Rappaport-Vassiliadis (MSRV) medium for 16 h at 42 degrees C. After enrichment, the total test time for the BEIA was 1.5 h. The analytical sensitivity of the BEIA ranged from 6.0 x 10(2) CFU/ml to 1.2 x 10(5) CFU/ml in MKTT and from 1.4 x 10(5) to 2.3 x 10(6) CFU/ml in MSRV using six Salmonella serovars prevalent in Canada. With enrichment cultures, the BEIA detected 1 CFU of Salmonella Typhimurium and Salmonella Enteritidis in 25 ml of chicken rinses. Representative strains of 10 Salmonella serovars were detected, and cross-reactivity was not observed with 25 non-Salmonella foodborne bacteria. The BEIA performance was assessed by testing 420 poultry samples, which were analyzed in parallel with the standard MSRV culture method. The BEIA detected 117 (27.88%) Salmonella-positive samples, whereas the standard MSRV culture method detected 124 (29.5%). The BEIA had a sensitivity of 64.5% and a specificity of 87.5% compared to the standard MSRV culture method. However, similar specificities and sensitivities were obtained when the standard MSRV culture method was compared to the BEIA (sensitivity = 68.4% and specificity = 85.5%). Neither method detected 100% of the Salmonella found in the samples tested, and statistical analyses indicated no significant difference between the two methods. In summary, the BEIA offers another alternative for the detection of Salmonella, with the additional advantage of providing a 24-h test for detecting Salmonella in chicken carcass rinses. The results obtained in this research indicate that tests are still needed for the isolation and detection of Salmonella that will establish the true prevalence of Salmonella in chicken samples.     

Detection of wheat as an allergenic substance in food by a nested PCR method

A nested PCR method was developed for the detection of DNAs extracted from allergenic substances (here, wheat) in food. Because of DNA fragmentation, detection of wheat-specific DNA extracted from food, such as retort pouch food, is very difficult. Therefore, to improve the sensitivity of detection, a nested PCR primer pair (Wtr01NE2-5' and Wtr10NE5-3': amplicon size 97 bp) was newly designed within the region of the PCR products amplified by the official Japanese primer pair (Wtr01-5' and Wtr10-3'; amplicon size 141 bp) for wheat. Genomic DNAs of seven kinds of commercial processed foods containing wheat, wheat flour and three kinds of wheat flours pressure-heated at 100, 121 and 131 degrees C were extracted with a commercial ion-exchange type kit by modifying the Japanese official method. The nested PCR method involved two PCR procedures. First, PCR was performed by varying both the PCR reagents and cycling conditions of the Japanese official method. Second, PCR was performed using the first PCR products diluted 200-fold with TE buffer. The Japanese official method enabled detection of only four of the seven kinds of foods and three of the four kinds of flours (one sample was just a trace), while the nested PCR method detected all seven foods and all four flours. Investigation of the detectability of the four kinds of wheat flours depending on the size of the amplified fragment using five primer pairs showed that its size must be kept to less than approximately 100 bp. The nested PCR method significantly improved the sensitivity of detection of wheat-specific DNA.