Development and application of a sensitive,rapid, and reliable immunomagnetic separation-PCR detection method for Cronobacter spp.

Cronobacter spp. have been linked to clinical cases of infection in both adults and infants. Enrichment of Cronobacter spp. before detection has been necessary but is quite time consuming. Hence, we sought to develop an immunomagnetic separation (IMS) PCR method that could shorten the time of enrichment before the detection of Cronobacter spp. The polyclonal antibody used in this immunomagnetic separation was prepared based on the outer membrane protein A of Cronobacter sakazakii China Center of Industrial Culture Collection 21560 and had high specificity to the target. The primers used in the IMS-PCR method also showed high specificity. The detection limit of IMS-PCR for pure C. sakazakii culture was 5.2 × 10² cfu/mL. Cronobacter sakazakii in artificially contaminated powdered infant formula (PIF) was also detected at a detection limit of 5.2 × 10² cfu/mL. After 8 h of enrichment, the detection limit in PIF was lower than 5.2 × 10¹ cfu/mL. An interference test using Escherichia coli in artificially contaminated PIF showed that the IMS-PCR method developed in this study had a good ability to resist interference. Finally, the IMS-PCR method was applied to the detection of Cronobacter in food samples and was shown to be reliable. Thus, this newly developed IMS-PCR detection method was quite sensitive, rapid, and reliable and could be applied to the detection of Cronobacter in foods.     

Biotin exposure–based immunomagnetic separation coupled with sodium dodecyl sulfate,propidium monoazide,and multiplex real-time PCR for rapid detection of viable Salmonella Typhimurium,Staphylococcus aureus,and Listeria monocytogenes in milk

In this study, we established a rapid and sensitive method for the detection of viable Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes in milk using biotin-exposure-based immunomagnetic separation (IMS) combined with sodium dodecyl sulfate (SDS), propidium monoazide (PMA), and multiplex real-time PCR (mRT-PCR). We used IMS to lessen the assay time for isolation of target bacteria. We then optimized the coupling conditions and immunomagnetic capture process. The immunoreaction and incubation times for 5 μg of mAb coupled with 500 μg of streptavidin-functionalized magnetic beads using a streptavidin-biotin system were 90 and 30 min, respectively. Treatment with SDS-PMA before mRT-PCR amplification eliminated false-positive outcomes from dead bacteria and identified viable target bacteria with good sensitivity and specificity. The limit of detection of IMS combined with the SDS-PMA-mRT-PCR assay for the detection of viable Salmonella Typhimurium, Staph. aureus, and L. monocytogenes in spiked milk matrix samples was 10 cfu/mL and remained significant even in the appearance of 10⁶ cfu/mL of nontarget bacteria. The entire detection process was able to identify viable bacteria within 9 h. The combination of biotin-exposure-mediated IMS and SDS-PMA-mRT-PCR has potential value for the rapid and sensitive detection of foodborne pathogens.     

Evaluation of an immunomagnetic separation-real-time PCR assay for the rapid detection of Salmonella in meat

The aim of this study was the comparison of an immunomagnetic separation (IMS)-real-time PCR assay for the detection of Salmonella with the cultural reference method according to and 35 of the German Law on Food and Commodities (LMBG, L 00.00.20:1998). The IMS-real-time PCR assay includes a nonselective preenrichment step, an IMS, DNA extraction, as well as DNA purification followed by hybridization probe-based real-time PCR analysis. An accurate comparability was achieved, because both methods analyzed the same preenrichment. The evaluation was carried out using both artificially and naturally contaminated meat samples. The IMS-real-time PCR assay provides a result after 12 to 13 h. Compared with the reference method and regarding artificially contaminated meat samples, the IMS-real-time PCR assay achieved a specificity of 80% (false-positive rate of 20%) and a sensitivity of 100% (false-negative rate of 0%). The relative accuracy was 94%. The detection limit of both methods was 10 CFU/25 g. The concordance index kappa, which defines the statistical accordance, was 0.85 and indicated the agreement of both methods on statistical criteria. Compared to the reference method and analyzing naturally contaminated meat samples (n = 491), the IMS-real-time PCR assay showed a specificity of 99.3% (false-positive rate of 0.7%) and a sensitivity of 83.7% (false-negative rate of 16.3%). The relative accuracy was 98%. The concordance index kappa had a value of 0.87 and highlighted the statistical agreement of both methods. In conclusion, the IMS-real-time PCR assay is suitable as specific, sensitive, and rapid screening method for the detection of Salmonella from meat.     

Combination of immunomagnetic separation and polymerase chain reaction for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in food samples.

A method that combined the immunomagnetic separation (IMS) technique and the multiplex polymerase chain reaction (PCR) method (i.e., the IMS-mPCR method) was developed for simultaneous detection of Listreria monocytogenes and Salmonella spp. in food samples. When only the multiplex PCR method was used, it was found that if cell numbers of each of the two target organisms (L. monocytogenes and Salmonella spp.) were above the detection limit, but differed by more than 2 logs-e.g., n x 10(7) to n x 10(4) or n x 10(6) to n x 10(3)--the organism presenting the lower numbers might go undetected. Following the enrichment step with universal preenrichment (UP) broth, if an IMS method using equal quantities of anti-Listeria and anti-Salmonella immunomagnetic beads was performed prior to PCR, both pathogens could be detected unambiguously. Such results could be obtained for target organisms in food samples, such as milk, dairy, and meat products, if similar enrichment and IMS steps were performed prior to PCR.     

Detection of Salmonella in food over 30 h using enrichment and polymerase chain reaction

A 30-h method for the detection of Salmonella spp. in food was developed. The method involved preenrichment in buffered peptone water for 6–8 h, immunomagnetic separation (IMS) using Dynabeads^®anti-Salmonella , selective enrichment in Rappaport-Vassiliadis broth for 16–18 h, lysis of bacterial cells in sodium dodecylsulfate and NaOH solution at 95°C, and the polymerase chain reaction (PCR) using primers ST11 and ST15. The detection limit of the method was 10⁰cfu 25g⁻¹, as determined by the analysis of food samples artificially contaminated with S. enteritidis. When the reference material containing on average 5 cfu of S. panama was used for the artificial contamination, 3 out of 22 food samples were found to be false-negative. When the method was evaluated in comparison with the standard ISO 6579 method on 42 possibly naturally-contaminated food samples, one sample was found positive by the 30-h method, one sample was found positive by the ISO-method, and two samples were found positive by both methods. The developed method proved rapid, but produced a non-zero level of false-negative results.     

免疫磁球捕获-PCR检测牛奶中金黄色葡萄球菌的研究

对免疫磁球捕获-PCR（IMS-PCR）检测牛奶中金黄色葡萄球菌进行了初步研究。优化了免疫磁球制备参数,确定了金黄色葡萄球菌免疫磁球对目标菌的结合时间。研究结果显示,当纯菌浓度为101¹04CFU/m L水平时,金黄色葡萄球菌免疫磁球对目标菌的捕获率大于80%。通过对目标菌和非目标菌的检测,IMS-PCR检测方法显示了很强的特异性;在纯培养、无需增菌情况,IMS-PCR检测方法检测限为104CFU/m L;牛奶中的金黄色葡萄球菌经磁分离后增菌2h用PCR检测,可检测出104CFU/m L的金黄色葡萄球菌。      

Combined PCR and slot blot assay for detection of Salmonella and Listeria monocytogenes

Detection of Salmonella typhimurium and Listeria monocytogenes by the polymerase chain reaction (PCR) assay coupled with slot blot detection was investigated in this study. After being extracted from diluted bacterial culture with the extraction buffer, bacterial DNA was subjected to PCR. The slot blot assay was optimized and used to detect PCR products. The lowest detection level of this method was 10(3) cfu/ml in the original culture media for both pathogens, or 5 bacterial cells in the PCR reaction. Combined with immunomagnetic separation (IMS) to separate and concentrate bacteria from samples, the detection limit could be 40 cfu/ml of bacteria from milk samples. The whole detection procedure was completed within 7 h. After multiplex PCR (amplification of DNA from two different bacteria in the same PCR tube) and slot blot, a detection level of 10(3) cfu/ml was achieved in the simultaneous detection for both pathogens, which was similar to that of individual detection for each pathogen. The combination of PCR and slot-blot seems to be highly sensitive and time-efficient, and is therefore promising for routine use in the detection of Salmonella and L. monocytogenes in food samples such as milk.     

Microbial flora of technological interest in raw ovine milk during 6°C storage

Changes in the microbial flora of ovine milk of medium hygienic quality (initial mean total plate count 3.3  ×  10⁵ cfu/mL) were studied throughout refrigerated storage at 6°C. Total plate counts after 48 h of refrigerated storage (mean count 4.6  ×  10⁵ cfu/mL) were under the current standards in the European Union (EU) for raw ovine milk to be used for cheesemaking after heat treatment. However, after 96 h, mean total plate counts (1.6  ×  10⁷ cfu/mL) were above the standards. Lactococci were found at levels higher than Pseudomonas spp. in milk freshly drawn (54.5% and 3.5% of total plate count, respectively) and after 96 h at 6°C (47.9% and 33.9% of total plate count, respectively). Lactobacilli, enterococci, coliforms and thermodurics were found at values lower than lactococci and Pseudomonas spp., before and after refrigerated storage ( ≤  0.08% of the total plate count). A significant growth ( P  < 0.001) was detected for mesophiles, Pseudomonas spp. and lactococci after 96 h of refrigeration at 6°C; however, thermodurics, coliforms, lactobacilli and enterococci, showed no significant increase ( P  > 0.05). Presumptive Escherichia coli (β-glucuronidase-positive) underwent a decrease throughout storage at 6°C.     

CONCURRENT DETECTION OF ESCHERICHIA COLI O157:H7 AND SALMONELLA FROM A SINGLE ENRICHMENT IN 24 H

The objective of this study was to determine if a single assay protocol could result in the concurrent detection of Escherichia coli O157:H7 and Salmonella from a single sample grown in a single enrichment in 24 h. Twenty-five and 375 g of ground beef nonfat dry milk, and dry pet food samples were seeded with low (10 cfu/sample) and high (100 cfu/sample) levels ofE. coli O157:H7 and Salmonella cultures and incubated at 35 and 41C for 18 h for nonselective preenrichment. Incubated samples were analyzed by immunomagnetic separation (IMS) following a 6 h incubation for selective enrichment at 37C using M-broth and enzyme linked immumosorbent assay (ELISA). Depending on the food samples and the inoculation level, the minimum concurrent detection level of E. coli O157:H7 and Salmonella was <1 cfu/g in the samples at the competitor flora level of 10⁵ cfu/g or less in ground beef samples, but in other cases of higher competitor loads and low target inoculations E. coli O157:H7 could not be detected in the presence of the Salmonella.     

免疫磁捕获-荧光定量PCR快速检测食品中沙门氏菌

建立了免疫磁珠分离（Immunomagnetic separation,IMS）联合荧光定量PCR（Real-time PCR）技术准确、快速检测食品中沙门氏菌（Salmonella spp）的方法。采用亲和纯化的羊抗沙门氏菌抗体与Dynabeads M-280磁珠制备沙门氏菌免疫磁珠,优化反应条件,建立免疫磁珠分离方法。同时根据沙门氏菌特异性ttr基因,建立Real-time PCR体系,并检测其特异性及敏感性。结果显示,沙门氏菌可产生荧光信号,而其他细菌均未见明显荧光信号。利用所建立的免疫磁捕获-荧光定量PCR（IMS-real-time PCR）方法可以检测初始含菌量最低为6.5CFU/25g的食品样品,全过程需时约8h。150份实际食品样品中,IMS-real-time PCR方法检出27份阳性样品,免疫磁珠联合XLT4平板培养法检出18份阳性样品,传统国标法检出14份阳性样品。      

Some virulence properties and characterization of motile Aeromonas species from milk and white cheese

Three hundred and thirty-eight samples of milk and milk products in Ankara were screened for the presence of motile Aeromonas species. The overall frequency of aeromonads in these samples was 27%. As expected, raw milk samples were more contaminated with aeromonads than were other products. Sixty-five (49.2%) of the 132 bulk raw milk samples were contaminated with aeromonads. In 35 of these samples, populations of aeromonads from 1.5 × 10² to 3.0 × 10³ cfu/mL were counted in ampicillin dextrin agar (ADA). Ten (40%) of the 25 raw milk samples sold in the street were contaminated with aeromonads. In eight of these samples, 1.2 × 10²−3.0 × 10³ cfu/mL were counted. Five (16%) of the 31 pasteurized milk and 12 (8%) of the 150 white cheese samples were contaminated with Aeromonas spp., but no countable aeromonads population was noted in ADA. The incidence of A. hydrophila, A. sobria and A. caviae in all samples was found to be 90.2%, 4.3% and 5.4%, respectively. The majority of the strains identified as A. hydrophila and A. sobria were able to produce haemolysin, protease and DNase, while strains identified as A. caviae were only positive for DNase. All isolated Aeromonas species ( A. hydrophila, A. sobria and A. caviae ) were positive for uptake of Congo red dye. Nevertheless, only strains identified as A. hydrophila and A. sobria showed a high rate of positive results when tested for the production of the Voges–Proskauer reaction and lysine decarboxylase. The results of this work show that motile Aeromonas spp., especially A. hydrophila , are frequently found in these samples. As these products are usually commonly consumed in Ankara, they can pose a risk especially for children, the elderly and immunocompromised individuals.     

肉及肉制品中沙门氏菌活细胞的PCR检测研究

将荧光染料叠氮溴化丙锭(propidium monoazide,PMA)与普通聚合酶链式反应(polymerase chain reaction,PCR)结合,通过对PMA的曝光时间、浓度进行优化,确定PMA-PCR区别死活细胞的最佳条件,并制作活细胞定量标准曲线,建立肉及肉制品中沙门氏菌活细胞的PMA-PCR检测方法。结果表明：使插入死细胞DNA中的PMA活化并且光解溶液中游离PMA的最佳曝光时间为15min;不抑制沙门氏菌活细胞DNA扩增的最大PMA质量浓度为10μg/mL;完全抑制热致死细胞DNA扩增的最小PMA质量浓度为4μg/mL。经PMA处理,含有不同比例的沙门氏菌热致死细胞和活细胞的混合液中活的沙门氏菌能够通过PCR被选择性的检测,最小检测限为20CFU/PCR。而且,经研究发现在20～2×105CFU/PCR范围内,电泳条带相对荧光强度与活细胞数的对数具有线性关系。采集30份肉及肉制品样品,利用PMA-PCR方法检测出两份生肉样品中存在沙门氏菌,经过6h的富集培养后的活菌浓度分别为2.5×103CFU/mL和3.4×103CFU/mL。     

Rapid detection of Salmonella in milk by combined immunomagnetic separation-polymerase chain reaction assay

During the past few years, milk has presented a risk of Salmonella contamination; it has been implicated as the cause in several outbreaks of salmonellosis. Because conventional detection methods require 5 to 7 d for completion and involve several subcultivation stages followed by biochemical and serological tests, rapid and sensitive methods have been sought, mainly at the DNA level. Therefore, a study including milk samples was conducted to evaluate the performance of a combination of 2 techniques—immunomagnetic separation and polymerase chain reaction (PCR)—for the detection of Salmonella. The 16-, 14-, 12-, 10-, and 8-h nonselective pre-enrichment steps before immunomagnetic separation and the high-pure DNA preparation method before PCR were used in a combined assay. Milk samples, which were found to be Salmonella-negative by a reference method, were first inoculated with Salmonella Enteritidis. Next, the shortest pre-enrichment time that is required for detection of 1 or 10 cfu of Salmonella/mL by combined immunomagnetic separation-PCR assay was found by using 16-, 14-, 12-, 10-, and 8-h incubation periods. The detection limit using a 16-, 14-, or 12-h nonselective pre-enrichment was 1 to 10 cfu/mL. However, the sensitivity decreased to 10¹ and 10² cfu/mL, respectively, when 10- and 8-h pre-enrichments were used. This assay, in conjunction with a 12-h pre-enrichment, proved to be rapid (overall 16 h) and sensitive (1-10 cfu/mL) for the detection of Salmonella in milk samples and promising for routine use in the detection of Salmonella in milk.     

PCR assay for detection of the E. coli O157:H7 eae-gene and effect of the sample preparation method on PCR detection of heat-killed E. coli O157:H7 in ground beef

A PCR assay targeting the 3' end of the eae-gene of E. coli O157:H7 has been developed. It was shown to be specific for the E. coli O157:H7 eae-gene. Sensitivity of the PCR assay was 1 pg DNA or 10(3) cfu per PCR reaction. Subsequently, a study was conducted to examine the effect of the food matrix and the sample preparation method on PCR detection of non-viable cells using heat-killed E. coli O157:H7 in ground beef as a model system. Inoculated ground beef samples were subjected to either selective enrichment or immediately prepared for PCR analysis. Four sample preparation methods were applied: centrifugation, buoyant density centrifugation (BDC), immunomagnetic separation (IMS), and chelex extraction. Production of false-positive results due to amplification of the DNA of dead cells occurred if the number of heat-killed cells exceeded 10(8) cfu/g. For inocula <10(8) cfu/g, PCR results for heat- killed E. coli O157:H7 cells depended upon the sample preparation method. It was shown that the inclusion of two washings steps of the bacterial pellet before DNA extraction was the critical factor for elimination of false-positive results due to heat-killed cells in the centrifugation and BDC procedure. IMS did not produce false-positive results due to heat-killed cells (two washing steps were included in the IMS procedure). With the chelex-extraction method, false-positive results due to heat-killed cells were obtained. An effect of the ground beef food matrix on the presence of amplifiable DNA was noted.     

免疫磁珠-环介导等温扩增快速检测牛肉中的鼠伤寒沙门氏菌与金黄色葡萄球菌

建立免疫磁珠分离（immunomagnetic separation,IMS）联合环介导等温扩增（loop-mediated isothermal amplification,LAMP）技术检测牛肉中鼠伤寒沙门氏菌与金黄色葡萄球菌的方法。用生物素标记的鼠伤寒沙门氏菌抗体和生物素标记的金黄色葡萄球菌菌体蛋白抗体对链霉亲和素磁珠进行功能化,从牛肉中捕获和分离目标致病菌。结果表明：经优化后,每毫克磁珠与10 μL鼠伤寒沙门氏菌抗体偶联,400 μL鼠伤寒沙门氏菌免疫磁珠在45 min内对104 CFU/mL鼠伤寒沙门氏菌的捕获率为52.16%;每毫克磁珠与6 μL金黄色葡萄球菌抗体偶联,300 μL金黄色葡萄球菌免疫磁珠在30 min内对104 CFU/mL金黄色葡萄球菌的捕获率为56.80%;建立的IMS-LAMP方法特异性高,对牛肉中鼠伤寒沙门氏菌的检测灵敏度为1.2×103 CFU/mL,对金黄色葡萄球菌的检测灵敏度为4.4×104 CFU/mL;富集5 h后,鼠伤寒沙门氏菌的检测限可至1.2 CFU/mL,富集7 h后,金黄色葡萄球菌的检测限可降至4.4 CFU/mL。建立的IMS-LAMP方法用时短,灵敏度高,操作简单,可以有效检测牛肉中的鼠伤寒沙门氏菌和金黄色葡萄球菌。     

Rapid detection of Salmonella typhimurium in chicken carcass wash water using an immunoelectrochemical method

An immunoelectrochemical method coupled with immunomagnetic separation was developed for rapid detection of Salmonella Typhimurium in chicken carcass wash water. Samples of chicken carcass wash water were inoculated with Salmonella Typhimurium at different cell numbers. Possible nonspecified inhibitors in the wash water were minimized by filtration and centrifugation. An approximately 9.4% loss of Salmonella cells was found after filtration (P < 0.01). The samples were mixed with anti-Salmonella-coated magnetic beads (ASCMB) and alkaline phosphatase-labeled anti-Salmonella (APLAS) to form ASCMB-Salmonella-APLAS conjugates. The conjugates were separated from the solution using a magnetic separator and then incubated with phenylphosphate substrate to produce phenol. The number of Salmonella was determined by measuring the phenol concentration using an amperometric tyrosinase carbon paste electrode in a flow injection analysis system. Under optimized parameters (1 mM MgCl2, 0.2 microg/ml APLAS, and 1 mM phenylphosphate in pH 7.0 Tris buffer solution), Salmonella Typhimurium in chicken carcass wash water could be identified and enumerated within 2.5 h with a detection limit of 5 x 10(3) CFU/ml. A linear relationship on a log-log scale was found between Salmonella cell number and the peak current ratio for Salmonella concentrations ranging from 10(3) to 10(7) CFU/ml (R2 = 0.963). The peak currents of multibacteria samples, containing Salmonella Typhimurium, Listeria monocytogenes, and Campylobacter jejuni, were not significantly different from Salmonella-only samples (P > 0.01).     

GROWTH INHIBITION OF CLOSTRIDIUM PERFRINGENS VEGETATIVE CELLS AND SPORES USING CHICKEN IMMUNOGLOBULIN Y

Egg yolk antibody (IgY) was isolated by the water dilution method from the egg yolk of chickens immunized with Clostridium perfringens vegetative cells and spores. Specific binding activity of IgY against C. perfringens vegetative cells and spores remained relatively high during the immunization period (up to 9 weeks). The titer of specific IgY against C. perfringens spores was 1.4-fold less than that of specific IgY against C. perfringens vegetable cells. The specific IgY powder (10 µg/mL) was found to inhibit the growth of C. perfringens vegetative cells or C. perfringens spores in a liquid medium. The difference of C. perfringens vegetative cell growth between the treatment and control groups was 8.9  ×  10⁶ colony forming units (cfu)/mL at 8 h of incubation and 9.95  ×  10⁷ cfu/mL at 24 h of incubation. Significant cfu reductions in C. perfringens spores were also observed with specific IgY powder at 24 h of incubation.

PRACTICAL APPLICATIONS

IgY antibody exerts an antimicrobial activity against pathogens by binding, immobilizing and consequently reducing or inhibiting the growth, replication or colony forming ability of pathogenic bacteria; thus, it is proved to be a viable alternative for antibiotics and preservatives. In this study, IgY against Clostridium perfringens can be used to replace chemical preservatives in food industries. Because IgY functions well at low temperature, it can be used to inhibit the growth of Clostridia which germinate in refrigerated storage conditions, thus preventing foodborne enterotoxicity caused by such bacteria. In practical applications, natural antimicrobial IgY antibody can be applied to meat products for the improvement of food safety.     

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.     

Improved Sample Preparation for Real-Time PCR Detection of Listeria monocytogenes in Hot-Smoked Salmon using Filtering and Immunomagnetic Separation Techniques

This work evaluated the application of filtration and immunomagnetic separation (IMS) as sample pretreatments for use in combination with real-time polymerase chain reaction (PCR) to detect and quantify Listeria monocytogenes in hot-smoked salmon. Salmon was artificially inoculated with L. monocytogenes at levels ranging from 8 × 10⁰ to 8 × 10⁵ cfu/g of sample, and homogenates obtained from these samples were filtered to recover bacterial cells without a pre-enrichment step. High recovery of bacterial cells was achieved using standard coffee filters. IMS significantly reduced the co-extraction of PCR inhibitors present in the samples to increase the assay sensitivity with regression line parameters applicable for quantification. The limit of detection and quantification were equal to 2 × 10¹–4 × 10¹ and 2 × 10² cfu/g of sample, respectively. The entire detection procedure could be completed within 3.5 h. This study demonstrated that coupling filtration and IMS with real-time PCR has contributed to improve the sensitivity of L. monocytogenes detection from hot-smoked salmon.