A double antibody sandwich enzyme-linked immunosorbent assay for the detection of Salmonella using biotinylated monoclonal antibodies

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed using monoclonal antibodies (MAbs) as a rapid, economical alternative to culture isolation procedures for detection of Salmonella. Four MAbs previously shown to react with Salmonella strains representing 18 different serogroups were evaluated as capture antibodies and, after biotinylation, as detection antibodies. One MAb (M183) was selected for use in the ELISA to capture and detect Salmonella antigens. The detection limit of the ELISA was evaluated using Salmonella enterica subspecies enterica serovar Typhimurium and various selective and nonselective Salmonella enrichment media. The highest detection limit (ca. 10(4) CFU/ml) was achieved using an enrichment broth containing brain heart infusion, yeast extract, sodium hydrogen selenite, and sodium cholate (BYSC) after preenrichment in buffered peptone water. The ELISA detected all Salmonella serovars tested, which included representative serovars of serogroups B, C, D, and E and gave negative results for all non-Salmonella species tested. Samples (106) from various sources, including fecal samples from humans and pigeons, chicken carcass rinses, chicken parts, feed, and the environment, were used to evaluate the performance of the ELISA. The ELISA had a specificity and sensitivity of 100 and 91%, respectively, and a kappa value of 0.93 relative to the culture methods. Such an ELISA has the potential to be used in the implementation of the pathogen reduction and hazard analysis critical control point systems as well as in clinical laboratories.     

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.     

通用多重不对称PCR结合寡核苷酸芯片检测7种食源性致病菌

应用通用多重不对称聚合酶链式反应(polymerase chain reaction,PCR)和寡核酸芯片技术建立一种同时检测7种常见食源性致病菌的方法。每种致病菌上游或下游引物5’端连接一段异源的共有序列。以荧光标记的该共有序列作为通用引物,与限制性特异引物经一步多重不对称PCR同时获得所有目标菌的单链标记靶序列,可被芯片上固定的特异性寡核苷酸探针捕获。通过芯片扫描、分析荧光信号完成检测。标准菌株检测结果证实,该方法可特异地检测单一和混合感染的目标菌,基因组DNA的检测灵敏度为0.1~1 pg。95份模拟污染和零售食品样本芯片检测结果与常规的分离与生化鉴定及荧光定量PCR结果一致。建立的寡核苷酸芯片方法可为快速、特异、灵敏及高通量地鉴定食源性致病菌提供一种有效的检测手段。     

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.     

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。     

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.     

Rapid, specific detection of Salmonella Enteritidis in pooled eggs by real-time PCR

An assay was developed for the specific detection of Salmonella Enteritidis in eggs with the use of an application of the fluorogenic 5' nuclease assay (TaqMan). In this assay, a segment of the gene sefA specific to Salmonella group D strains such as Salmonella Enteritidis was used. The amplification of the target gene products was monitored in real-time by incorporating a fluorescent dye-labeled gene-specific probe in the PCR reaction. This method correctly detected and distinguished Salmonella Enteritidis from nearly 50 of non-group D Salmonella and other non-Salmonella strains. Detection of the sefA gene was linear for DNA extracted from approximately 10(2) to 10(9) CFU/ml in phosphate-buffered saline and 10(3) to 10(8) CFU/ml in raw egg. In two trials, when applied to detection of Salmonella Enteritidis in homogenized egg pools and compared with conventional culture methods, the newly developed PCR method yielded a 100% correlation with results obtained by a conventional culture method. However, the PCR method required only 2 days, compared to the 5 days required by the culture method. The sensitivity of this assay was approximately less than 1 CFU/600 g of egg pool. The real-time PCR assay proved to be a rapid, highly sensitive test for detection and quantification of low concentrations of Salmonella Enteritidis in egg samples.     

High-pressure resistance variation of Escherichia coli O157:H7 strains and Salmonella serovars in tryptic soy broth, distilled water, and fruit juice

The effect of high pressure on the log reduction of six strains of Escherichia coli O157:H7 and five serovars of Salmonella enterica was investigated in tryptic soy broth, sterile distilled water, and commercially sterile orange juice (for Salmonella) and apple cider (for E. coli). Samples were subjected to high-pressure processing treatment at 300 and 550 MPa for 2 min at 6 degrees C. Samples were plated onto tryptic soy agar directly after pressurization and after being held for 24 h at 4 degrees C. At 300 MPa, little effect was seen on E. coli O157:H7 strains, while Salmonella serovars varied in resistance, showing reductions between 0.26 and 3.95 log CFU/ml. At 550 MPa, E. coli O157:H7 strains exhibited a range of reductions (0.28 to 4.39 log CFU/ml), while most Salmonella populations decreased beyond the detection limit (> 5-log CFU/ml reduction). The most resistant strains tested were E. coli E009 and Salmonella Agona. Generally, bacterial populations in fruit juices showed larger decreases than did populations in tryptic soy broth and distilled water. E. coli O157:H7 cultures held for 24 h at 4 degrees C after treatment at 550 MPa showed a significant log decrease as compared with cultures directly after treatment (P < or = 0.05), while Salmonella serovars did not show this significant decrease (P > 0.05). All Salmonella serovars tested in orange juice treated at 550 MPa for 2 min at 6 degrees C and held for 24 h showed a > 5-log decrease, while E. coli O157:H7 strains require a higher pressure, higher temperature, longer pressurization, or a chemical additive to achieve a 5-log decrease.     

An improved PCR primer pair based on 16S rDNA for the specific detection of Salmonella serovars in food samples

Salmonella serovars are some of the major bacterial pathogens that can cause sporadic cases and outbreaks of foodborne illness. Based on the sequence data in the V3 region of the 16S rRNA gene, two PCR primer pairs have been designed for the detection of all serovars of Salmonella. However, none of these primers were specific for Salmonella because complete sequence homology with certain non-Salmonella strains has been found within each of them. Thus, the specificities of these two primer pairs could not rely on only one of the two primers. In this study, we modified our previous 16SFI primer by extending one base at the 5' end and three bases at the 3' end. The modified primer, 16S-Sal, was designed with one or more mismatched bases near the 3' end of the primer annealing to the corresponding sequences of non-Salmonella strains. Such modification eliminates interference from Citrobacter freundii and Enterobacter cloacae as occurs with the 16SFI primer. When 16S-Sal and a degenerate primer, 16S-CCR, were used as a primer pair, detection specificity of Salmonella serovars was achieved. Because this primer pair was used for PCR detection of the salmonellae in food samples, such as whole milk and chicken meat, as low as 1 to 9 CFU/g (ml) of the food sample could be detected when a 8-h preculture step was performed prior to the PCR. For chicken meat, the endogenous microflora did not interfere with the PCR results.     

Probability of detection of Salmonella using different analytical procedures, with emphasis on subspecies diarizonae serovar 61:k:1,5,(7) [S. IIIb 61:k:1,5,(7)

A parameter for assessing qualitative methods is suggested: 'The probability of detection' has been used in this study to compare the efficiency of commonly used media for detecting Salmonella in mutton and faeces. Mutton and ovine faeces spiked with strains of Salmonella IIIb 61:k: 1,5,(7) and Salmonella Typhimurium were analysed and the performance of each combination of media was estimated. Extensive variation between serovars and strains of identical serovars were recorded. In meat, an inoculum of up to 10(6)/g was needed to detect salmonellae with 90% probability, while in faeces even 10(7)/g was not enough for some strains of S. IIIb 61:k:1,5,(7). All method combinations thus had a rather low efficiency. Based on our experiments, we recommend using a combination of the XLD medium and any of the three selective broths for detection of S. IIIb 61:k:1,5,(7) from mutton, while we recommend using the SC broth with any of the three plating media for detection from ovine faeces.     

Real-time multiplex SYBR green I-based PCR assay for simultaneous detection of Salmonella serovars and Listeria monocytogenes

A multiplex SYBR Green I-based PCR assay has been developed for simultaneous detection of Salmonella serovars and Listeria monocytogenes using a LightCycler. Primers were designed to amplify an 85-bp sequence from the gene encoding a fimbrinlike protein (fimI) of Salmonella Enteritidis and a 98-bp sequence from the hemolysin gene (hly) of L. monocytogenes. These primers allowed the amplification of PCR products having distinct melting temperature values, resulting in the formation of two distinct peaks representing the two targets. Background signals, resulting from primer-dimer formation in the late cycles of PCR, are eliminated through the acquisition of data at a high temperature (>75 degrees C), but several degrees lower than required for detection of the specific PCR products. A rapid and simple method for the extraction of bacterial genomic DNA from liquid culture, coupled with duplex PCR using LightCycler SYBR Green-based PCR assays, detected the presence of 2.5 cells and 1 cell of Salmonella serovars and L. monocytogenes, respectively, within an hour. Following overnight enrichment, target DNA was present in sufficient quantities in 1 microl of culture to enable direct detection with the LightCycler.     

沙门菌属特异性检测靶点碱基差异位点的识别及其血清型决定力

以美国国家生物技术信息中心(NCBI)已公布的28株沙门菌(14个血清型)全基因组序列为研究对象,对前期研究获得的7个沙门菌属特异性检测靶点在不同血清型间的单核苷酸多态性进行比较分析,结果表明,S9和S69为碱基差异位点最多的两个靶点,具有沙门菌分子血清分型的潜在能力。随后,通过分析S9和S69在沙门菌不同血清型菌株中的差异位点,分别绘制两个血清分型靶点的差异位点表,从而获得了这两个靶点同时用于14个沙门菌血清型的快速分子血清分型的差异位点组合。在这些组合中,同一血清型具有相同的差异位点,不同血清型可以通过差异位点得以区分。最后,采集食品样品192份,用国标方法获得疑似沙门菌21株;利用血清分子分型靶点S9和S69进行快速分型,并同时用传统玻片凝集反应鉴定方法进行验证,两种方法鉴定结果的符合率为100%。鉴定结果为:21株沙门菌共包括4个不同血清型,其中肠炎沙门菌10株、鼠伤寒沙门菌7株、鸡沙门菌2株、纽波特沙门菌2株。基因组序列分析结果和分离株分型结果均表明,沙门菌特异分子检测靶点S9和S69相结合具备沙门菌的分子血清分型能力,以差异位点表为基础建立的血清分型方法有望替代传统的玻片凝集血清分型这一繁杂步骤,从而降低沙门菌血清鉴定的时间与成本,为聚合酶链式反应技术应用于沙门菌分子血清分型提供新思路。     

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。     

Salmonella DNA adenine methylase mutants prevent colonization of newly hatched chickens by homologous and heterologous serovars

Salmonella mutants lacking DNA adenine methylase (Dam) are highly attenuated for virulence and confer protection against oral challenge with homologous and heterologous Salmonella serovars in mice and chicken broilers. To determine whether vaccines based on Dam are efficacious in preventing early colonization of newly hatched chickens, a Salmonella typhimurium Dam(-) vaccine was evaluated for the protection of chicks against oral challenge with homologous and heterologous Salmonella serovars. Vaccination of chicks elicited protection 2 and 6 days post-challenge as evidenced by a significant reduction in colonization of the gastrointestinal tract (ileum, cecum and feces) and visceral organs (spleen and bursa) when challenged with homologous S. typhimurium. Moderate protection was observed following challenge with heterologous S. enteritidis and Salmonella O6, 14, 24:e, h-monophasic) serovars. These data suggest that Salmonella Dam mutant strains conferred cross-protection, presumably via competitive exclusion mechanisms that prevent superinfection of chicks by other Salmonella strains. Such protection may reduce pre-harvest Salmonella contamination in poultry, decreasing the potential for food-borne transmission of this pathogen to humans.     

Detection of multiple antibiotic-resistant Salmonella enterica serovar Typhimurium DT104 by phage replication-competitive enzyme-linked immunosorbent assay

A phage replication-competitive enzyme-linked immunosorbent assay (PR-cELISA) was developed for the detection of multiple antibiotic-resistant Salmonella Typhimurium DT104. In the PR-cELISA procedure, a phage, BP1, was inoculated into a log-phase bacterial culture at a ratio of 1:100. After a 3-h incubation of the mixture, BP1 replication was measured by cELISA based on the competitive binding between BP1 and biotinylated BP1 to Salmonella Typhimurium smooth lipopolysaccharide. Among the 84 Salmonella strains and 9 non-Salmonella strains that were tested by PR-cELISA, BP1 detected 39 of 40 Salmonella Typhimurium strains, 2 of 10 Salmonella non-Typhimurium somatic group B strains, and 5 of 18 Salmonella somatic group D1 strains. With the addition of chloramphenicol to the culture medium, PR-cELISA detected all 27 multiple antibiotic-resistant Salmonella Typhimurium DT104 and none of the other Salmonella strains or non-Salmonella strains tested. The results demonstrated that PR-cELISA has potential applications for the detection of multiple antibiotic-resistant Salmonella Typhimurium DT104.     

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.     

Validated PCR assay for the routine detection of Salmonella in food

We developed a rapid and reliable PCR assay with genus-specific primers for the detection of Salmonella in food samples. With these primers, no primer-specific amplicons were detected when challenged with cultures of microorganisms other than salmonellae, and positive results, i.e., Salmonella-specific bands, were obtained with pure cultures of all 125 Salmonella isolates tested, which represented 100 serovars. The PCR assay was optimized using both pure cultures and artificially inoculated food samples. The assay results were compared with those of the Australian standard culture methods, using more than 500 "naturally" contaminated food samples, over a period of 9 years. Food samples were subjected to nonselective preenrichment in buffered peptone water followed by selective enrichment in Rappaport Vassiliadis (RV) broth and mannitol selenite cystine (MSC) broth. A simple sample preparation method was developed based on concentrating bacterial cells from 1 ml of RV or MSC broths. The PCR results were in perfect agreement with the results of the standard culture methods; no false-positive or false-negative results were obtained. However, the PCR assay was extremely rapid, and results could be obtained within 4 h of testing of enrichment broths.     

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.     

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.     

Preliminary evaluation of flow-through immunocapture followed by real-time PCR for the detection of Salmonella serovars on tomato surfaces within 8 hours

This study reports a preliminary evaluation of flow-through immunocapture (FTI) followed by real-time PCR (FTI-PCR) for the detection of Salmonella serovars on tomato surfaces within 8 h. The FTI-PCR method was compared with real-time PCR, direct plating of FTI beads on xylose lysine desoxycholate (XLD), and the conventional culture method for Salmonella found in the U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM). Unwaxed green tomatoes were spot inoculated with a five-serovar Salmonella cocktail on smooth surfaces at levels of 10(0) to 10(4) CFU per tomato and washed in lactose broth (LB) using a shake-rub method. The resulting LB rinse was incubated at 37 degrees C for 4 h prior to analysis by FTI-XLD, real-time PCR, or FTI-PCR and for 24 h as the first step in the BAM Salmonella culture method. For FTI-XLD, the observed lowest detection level (LDL) was 4.6 x 10(1) CFU per tomato. There was no significant difference in performance between the FTI-XLD method and the BAM Salmonella culture method (P > 0.05); however, the FTI-XLD method reduced the overall assay time by 48 h. For real-time PCR and FTI-PCR, the observed LDLs were 4.6 x 10(1) and 9.2 x 10(0) CFU per tomato, respectively. The FTI-PCR method was superior to the BAM Salmonella culture method (P < 0.05) for the detection of Salmonella serovars on tomato surfaces and was completed within 8 h.