Spiked Shrimp Samples Research Articles

Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification

ObjectiveTo establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP). MethodsWe established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD). ResultsCRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10-18 M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 102 CFU/g. ConclusionThe novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.

Rotary Valve-Assisted Fluidic System Coupling with CRISPR/Cas12a for Fully Integrated Nucleic Acid Detection.

Of late, many nucleic acid analysis platforms have been established, but there is still room for constructing integrated nucleic acid detection systems with high nucleic acid extraction efficiency, low detection cost, and convenient operation. In this work, a simple rotary valve-assisted fluidic chip coupling with CRISPR/Cas12a was established to achieve fully integrated nucleic acid detection. All of the detection reagents were prestored on the fluidic chip. With the aid of the rotary valve and syringe, the liquid flow and stirring can be precisely controlled. The nucleic acid extraction, loop-mediated isothermal amplification (LAMP) reaction, and CRISPR detection could be completed in 80 min. A clean reservoir and an air reservoir on the fluidic chip were designed to effectively remove the remaining ethanol. With Vibrio parahaemolyticus as the targets, the detection sensitivity of the fluidic chip could reach 3.1 × 101 copies of target DNA per reaction. A positive sample could be sensitively detected by CRISPR/Cas12a to produce a green fluorescent signal, while a negative sample generated no fluorescent signal. Further, the fluidic chip was successfully applied for detection of spiked shrimp samples, which showed the same detection sensitivity. A great feasibility for real-sample detection was showed by the fluidic chip. The proposed detection platform did not need expensive centrifugal instruments or pumps, which displayed its potential to become a powerful tool for food safety analysis and clinical diagnostics, especially in the resource-limited areas.

A reversible valve-assisted chip coupling with integrated sample treatment and CRISPR/Cas12a for visual detection of Vibrio parahaemolyticus

Vibrio parahaemolyticus (V. parahaemolyticus) is regarded as a major cause of seafood-associated illnesses, which has aroused widespread public concern. Here, a rapid and convenient detection method for V. parahaemolyticus detection was established by a reversible valve-assisted chip coupling with CRISPR/Cas12a. With optimized lysis buffer, loop mediated isothermal amplification (LAMP) reagents and CRISPR reagents, the whole detection process from sampling to results could be finished within 50 min. The structure of chip was simple and the cost was low. By relying on three reversible rotary valves and the rotation direction-dependent Coriolis pseudo force, the flow order of liquid and the direction of liquid flow could be precisely controlled. The LAMP amplicons were specifically and sensitively identified by CRISPR/Cas12a. Positive amplification would produce green fluorescent signal while negative amplification generated no fluorescent signal, which could be clearly distinguished by the naked eye. With 600 μL of samples processed, the limit of detection (LOD) for both pure cultured V. parahaemolyticus or spiked shrimp samples could achieve 30 copies/reaction. These illustrated the established method displayed great feasibility for real samples detection. In the future, the chip could also combine with other amplification reactions, like PCR or recombinase polymerase amplification reaction (RPA), to conduct detection by changing the corresponding lyophilized amplification reagents. Overall, the proposed detection platform displays great potential for food safety analysis and clinical diagnostics, especially in resource-limited areas.

Nanoparticle-based lateral flow biosensor combined with multiple cross displacement amplification for rapid, visual and sensitive detection of Vibrio cholerae.

Vibrio cholerae is an important human pathogen that is responsible for cholera, a severe acute watery diarrhea. In the current study, a multiple cross displacement amplification (MCDA) coupled with amplicon detection by chromatographic lateral flow biosensor (LFB) method (MCDA-LFB) was successfully established and evaluated for the identification of V. cholerae. A set of 10 primers was designed specifically to recognize 10 different regions of the V. cholerae-specific gene ompW. The optimized time and temperature conditions for the MCDA were 30 min and 63°C, respectively. The MCDA-LFB assay correctly identified 31 strains of V. cholerae but did not detect 13 non-cholerae Vibrio strains and 30 non-Vibrio strains. The sensitivity of MCDA-LFB for target pathogen detection in pure culture was 10 fg per reaction. In the case of spiked shrimp samples without enrichment, the limit of detection was 4.1 CFUs per reaction or equivalent to 4.1×102 CFU g-1. The whole process, including shrimp homogenates processing (30 min), MCDA reaction (30 min) and results reporting (2 min), could be finished within 65 min. These results show that this assay is suitable for the rapid, sensitive and specific detection of V. cholerae in food, environmental and clinical samples.

Development of Duplex Loop‐Mediated Isothermal Amplification (dLAMP) Combined with Lateral Flow Dipstick (LFD) for the Rapid and Specific Detection of Vibrio vulnificus and V. parahaemolyticus

AbstractLoop‐mediated isothermal amplification (LAMP) is a rapid, specific, and effective DNA amplification assay. In this study, a duplex LAMP (dLAMP) assay combined with chromatographic lateral flow dipstick (LFD) was developed for the simultaneous identification of Vibrio vulnificus and V. parahaemolyticus. Each primer set that comprised F3, B3, FIP, and BIP recognized the V. vulnificus rpoS gene and the V. parahaemolyticus tlh gene. Digoxigenilated and biotinylated LAMP amplicons of V. vulnificus and V. parahaemolyticus, respectively, were hybridized with corresponded fluorescein isothiocyanate (FITC)‐labeled probes for LFD detection. The optimum conditions were 90 min at 63°C. The dLAMP–LFD had high specificity to V. vulnificus and V. parahaemolyticus and showed no cross‐amplification with 59 isolates of other bacteria. The detection limit of dLAMP–LFD with a pure culture of V. vulnificus was 2.2 × 104 CFU/mL, corresponding to 41 CFU per reaction, which was equal to that of duplex PCR (dPCR). In the case of V. parahaemolyticus, the detection limit of dLAMP–LFD with a pure culture was 2.2 × 103 CFU/mL, corresponding to 4.1 CFU per reaction, 100 times more sensitive than that of dPCR. In the spiked shrimp samples, the detection limit of dLAMP–LFD for V. vulnificus was 2.2 × 105 CFU/g, corresponding to 410 CFU per reaction, 10 times more sensitive than that of dPCR. In the case of V. parahaemolyticus, the detection limit of dLAMP–LFD in spiked shrimp samples was 2.2 × 104 CFU/g, corresponding to 41 CFU per reaction, 100 times more sensitive than that of dPCR. These results demonstrate that the simple dLAMP–LFD assay had high specificity and high sensitivity for the concurrent detection of V. vulnificus and V. parahaemolyticus.

Rapid multiplex polymerase chain reaction for simultaneous detection of Vibrio harveyi, V. parahaemolyticus, and V. vulnificus in pacific white shrimp (Litopenaeus vannamei)

Context: A comparatively small number of species, e.g., Vibrio parahaemolyticus and V. vulnificus , cause disease in both aquatic animals and humans. V. harveyi is marine animal pathogen and rarely causes infections in humans; however, it might become a reservoir of antibiotic-resistant bacteria forms and virulence genes. Aims: 1) to develop rapid multiplex polymerase chain reaction (PCR) assay for the simultaneous detection of V. harveyi, V. parahaemolyticus , and V. vulnificus by using vhhP2, tl , and rpoS genes as the respective target genes and 2) to evaluate specificity and determined detection of multiplex PCR technique. Materials and Methods: The multiplex PCR assay was developed and evaluated for specificity on 36 isolates of V. harveyi , 30 isolates of V. parahaemolyticus , and 14 isolates of V. vulnificus , along with other species of Vibrio and non- Vibrio bacterial isolates. Sensitivity of test was described as detection limit of pathogens in lowest amount of sample (CFU/mL or CFU/g) was determined by diluted DNA extracts of the pure cultures and spiked pacific white shrimp (Litopenaeus vannamei) samples Results: This developed multiplex PCR was proved as an accurate method, which was specific for three Vibrio species. The detection limits of V. harveyi , V. parahaemolyticus , and V. vulnificus in pure cultures and spiked shrimp samples ranged 1.05-4.8 × 10 3 CFU/mL and 1.9-7 × 10 4 CFU/g, respectively. Conclusions: This rapid multiplex PCR assay can decrease amount and process of sample preparation, which was time-consuming, and had preferable accuracy. This developed technique will be suitable and useful for food-borne pathogen detection in shrimp and horizontal gene transfer study among different Vibrio species in aquatic animals.

Surface molecularly imprinted polymers based on yeast prepared by atom transfer radical emulsion polymerization for selective recognition of ciprofloxacin from aqueous medium

ABSTRACTTo achieve selective recognition of water‐soluble ciprofloxacin (CIP), an effective method was developed for the preparation of surface molecularly imprinted polymers based on the yeast particles (yeast@MIPs) via atom transfer radical emulsion polymerization (ATREP). The reactions were carried out in the nontoxic and green emulsion system at room temperature, which was environment friendly with low energy consumption. In this study, the yeast, for the advantages of low cost, easily available source and abundant active groups on the cell wall, was selected as an ideal biological support substrate. The prepared yeast@MIPs was characterized by FT‐IR, SEM, TEM, EDS, and elemental analysis techniques. Batch mode adsorption studies were carried out to investigate the specific adsorption equilibrium, kinetics, selective recognition, and reuse ability of yeast@MIPs. The experimental static adsorption data of CIP on to yeast@MIPs were well‐described by Langmuir, Freundlich, and pseudo‐second‐order models. The maximum static adsorption capacity for CIP of yeast@MIPs was 18.48 mg g−1, and the adsorption equilibrium could be reached in 60 min. The selectivity coefficients for CIP relative to enrofloxacin, tetracycline, and sulfadiazine were 1.212, 2.002, and 10.65, which demonstrated CIP of high affinity and selectivity over three competitive antibiotics. In addition, the reusability of the material without obvious deterioration (8.52% loss) in performance was observed at least four repeated cycles. And the yeast@MIPs was used to determine CIP from spiked shrimp samples by HPLC analysis. These results showed that yeast was a well‐defined substrate and ATREP was a promising technique for the preparation of surface molecularly imprinted polymers targeting templates. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40310.

Multiplex PCR (Polymerase Chain Reaction) Assay for Detection of E. coli O157:H7, Salmonella sp., Vibrio cholerae and Vibrio parahaemolyticus in Spiked Shrimps (Penaeus monodon)

The coastal aquaculture mainly shrimps constitute major export sector in Bangladesh and is increasingly shaped by international trade conditions and by national responses to those stringent quality and safety standards. PCR based validated methods for detection of major bacterial pathogens in shrimp might be very useful tool for ensuring quality and safety standards of exportable shrimps. The objective of this study was to evaluate overall performance (sensitivity and specificity) of the multiplex PCR assay for detection of Vibrio cholerae, Vibrio parahaemolyticus, Salmonella sp. and Escherichia coli O157:H7 from spiked shrimp samples. The targeted genes were ompW for V. cholerae, tdh for V. parahaemolyticus, sefA for Salmonella spp. and hlyEHEC for E. coli O157:H7. The genomic DNA was extracted by using standard method and amplified accordingly. Sensitivity of the assay was tested by inoculating the shrimp homogenate with viable cells of laboratory references strains (target pathogens). The genes were amplified individually both from culture homogenate and spiked samples. Twenty different uniplex and multiplex PCR assay were performed; the results showed that the sensitivity and specificity of multiplex PCR are comparable to that of the results of uniplex PCR for the samples. DNA extracted from shrimp samples spiked with non-target pathogen (Bacillus cereus, Shigella flexneri and Staphylococcus aureus) yielded negative results.

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus

The current study was aimed to develop a loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay for rapid and specific detection of Vibrio parahaemolyticus. Biotinylated LAMP amplicons were produced by a set of four designed primers that recognized specifically the V. parahaemolyticus thermolabile haemolysin (tlh) gene followed by hybridization with an FITC-labelled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP-LFD method accurately identified 28 isolates of V. parahaemolyticus but did not detect 24 non-parahaemolyticus Vibrio isolates and 35 non-Vibrio bacterial isolates. The sensitivity of LAMP-LFD for V. parahaemolyticus detection in pure cultures was 120 CFU ml⁻¹. In the case of spiked shrimp samples without enrichment, the detection limit for V. parahaemolyticus was 1·8 x 10³ CFU g⁻¹ or equivalent to 3 CFU per reaction while that of conventional PCR was 30 CFU per reaction. The established LAMP-LFD assay targeting tlh gene was specific, rapid and sensitive for identification of V. parahaemolyticus. The developed LAMP-LFD assay provided a valuable tool for detection of V. parahaemolyticus and can be used effectively for identification of V. parahaemolyticus in contaminated food sample.

Supercritical fluid extraction in situ derivatization for simultaneous determination of chloramphenicol, florfenicol and thiamphenicol in shrimp

The applicability of supercritical fluid extraction in situ derivatization was investigated for determination of trace amounts of amphenicols (chloramphenicol, florfenicol and thiamphenicol) in shrimp. Quantification was performed by using electron-capture negative chemical ionisation-gas chromatography/mass spectrometry (NCI–GC/MS). The parameters of supercritical fluid extraction (addition of modifier, temperature, pressure, extraction time and extraction mode) and in situ derivatization (collection solvent and derivatization reagent) were varied with control. The optimum extractions were obtained using 600 μL ethyl acetate as a modifier for supercritical carbon dioxide with static extraction for 5 min, then dynamic extraction for 10 min at 25 MPa and 60 °C. The conditions for in situ derivatization were 200 μL N,O-bis(trimethylsilyl) trifluoroacetamide containing 1% trimethylchlorosilane in 20 mL ethyl acetate as collection solvent. The new method of supercritical fluid extraction in situ derivatization was found to be linear over the concentration range of 20–5000 pg/g, with detection limits ranging from 8.7 to 17.4 pg/g (using the selective ion monitoring mode), with a R.S.D. (relative standard deviation) less than 15.3% (n = 5). Analysis of spiked shrimp samples revealed that matrix had little effect on extraction. The results presented here indicate that supercritical fluid extraction in situ derivatization is for the trace analysis of amphenicol bacteriostats in shrimp samples.

Development of a Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of the tdh and trh Genes of Vibrio parahaemolyticus and Related Vibrio Species

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.

Rapid and sensitive detection of Vibrio cholerae by loop-mediated isothermal amplification targeted to the gene of outer membrane protein ompW

The present study was aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Vibrio cholerae. A set of five designed primers that recognized specifically the V. cholerae ompW gene was used. The optimized time and temperature conditions for the LAMP assay were 75 min at 65 degrees C, respectively. The LAMP method accurately identified 16 isolates of V. cholerae but did not detect 28 non-cholerae Vibrio isolates and 37 non-Vibrio bacterial isolates. The sensitivity of LAMP for V. cholerae detection in pure cultures was 2.2 x 10(3) CFU ml(-1) or equivalent to 8 CFU per reaction. In the case of spiked shrimp samples without enrichment, the detection limit for V. cholerae was 2.2 x 10(4) CFU g(-1) or equivalent to 20 CFU per reaction, while that of PCR was 100 CFU per reaction. The developed LAMP assay targeting ompW gene was rapid, specific and sensitive for V. cholerae detection. The developed LAMP assay appears to be precise, accurate and a valuable tool for detection of V. cholerae. This assay can replace laborious biochemical tests for the identification of V. cholerae in contaminated food sample.

Rapid separation and highly sensitive detection methodology for sulfonamides in shrimp using a monolithic column coupled with BDD amperometric detection

In this report, we aimed to extend our previous efforts toward the evaluation of sulfonamides (SAs) with a boron-doped diamond (BDD) electrode. We improved this method by reducing the analysis time using a monolithic column coupled with amperometric detection to determine seven sulfonamides (sulfaguanidine, sulfadiazine, sulfamethazine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline). Because of its rapid separation, low back-pressure and high separation efficiency compared to a particle-packed column, a monolithic column (100 mm × 4.6 mm) was used for sulfonamide separation. Chromatographic separation was performed in less than 8 min. The analysis was carried out using phosphate buffer (0.1 M, pH 3): acetonitrile: methanol in a ratio of 80:15:5 (v/v/v) as the mobile phase with a flow rate of 1.5 mL min −1. The optimal detection potential using hydrodynamic voltammetry was found to be 1.2 V versus Ag/AgCl. The method was applied to determine seven sulfonamides in shrimp after sample preparation by solid-phase extraction. The recoveries of the sulfonamides in spiked shrimp samples at 1.5, 5 and 10 μg g −1 were in the range of 81.7 to 97.5% with a relative standard deviation (R.S.D.) between 1.0 and 4.6%. Our methodology produced results that were highly correlated with HPLC–MS data. Therefore, we propose a method that can be used for the rapid, selective and sensitive evaluation of sulfonamides in contaminated food.

Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus

BackgroundVibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of Vibrio parahaemolyticus.ResultsThe assay provided markedly more sensitive and rapid detection of V. parahaemolyticus strains than conventional biochemical and PCR assays. The assay correctly identified 143 V. parahaemolyticus strains, but did not detect 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains. Sensitivity of the LAMP assay for direct detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples was 5.3 × 102 CFU per ml/g (2.0 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13–22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination.ConclusionThe LAMP assay is a sensitive, rapid and simple tool for the detection of V. parahaemolyticus and will facilitate the surveillance for control of contamination of V. parahaemolyticus in seafood.

Chemiluminescence enzyme immunoassay (CLEIA) for the determination of chloramphenicol residues in aquatic tissues

A competitive indirect chemiluminescence enzyme immunoassay (ic-CLEIA) for chloramphenicol (CAP) residues in shrimp has been developed. After optimization (incubation time, concentration of Tween-20, concentration of PBS and pH), the method gave a limit of detection of 0.01 ng/mL and a detection range of 0.03-23.7 ng/mL, with an ED(50) of 0.47 ng/mL. The method has been validated on spiked shrimp samples in terms of precision (intra- and interassay coefficient variations of less than 10% and 15%, respectively) and accuracy (mean recovery 95-123%). The assay performance is better than the ELISA method which is widely used to detect chloramphenicol and indicates that the CLEIA method can be used to test aquatic samples instead of ELISA.

Quantitative analysis of chloramphenicol residues in shrimp muscle tissues by Chemiluminescent enzyme immunoassay

A competitive indirect chemiluminescent enzyme immunoassay (ic-CLEIA) has been developed for the determination of chloramphenicol (CAP) residues in shrimp. After the optimisation of four physico-chemical parameters, i.e. incubation time, concentration of Tween-20, concentration of PBS and its pH, the method developed gave a limit of detection of 0.01 ng/ml and a detection range from 0.03 ng/ml to 23.7 ng/ml, with an ED<sub>50</sub> of 0.47 ng/ml. The developed method has been validated on spiked shrimp samples in terms of precision (intra- and interassay coefficient variations of less than 10% and 15%, respectively), and of accuracy (mean recovery from 95% to 123%). All these parameters being better than those of the ELISA method which is widely used to detect chloramphenicol, it may be suggested that the CLEIA method can be used to detect aquatic samples instead of ELISA.  

A LIQUID CHROMATOGRAPHIC FLUORESCENCE TECHNIQUE FOR ESTIMATING CRUDE OIL IN WATER, SEDIMENT, AND BIOLOGICAL MATERIAL

ABSTRACT This paper presents a method for the rapid detection of crude oil in the presence of large amounts of biological material. The method involves the use of high pressure liquid chromatography with chloroform as the solvent, an excitation wavelength of 403 nm, and an emission wavelength in the range of 418 nm. Of the five crude oils employed in this investigation, Saudi Arabian crude oil was the least responsive to the above technique and yet is still detectable in quantities of less than 1 µg. Reproducibility studies indicated that the percent error of repetitive injections of Saudi Arabian crude oil samples was 1.3%. Repetitive injections of spiked shrimp samples gave a percent error of 3.7%. This is excellent reproducibility for residue-type samples which undergo a number of manipulations during workup. Thus, this method is selective, sensitive, reproducible, and affords a rapid analysis.

Evaluation of Multiplex PCR System for Simultaneous Detection of <i>Escherichia coli</i> O157:H7, <i>Listeria monocytogenes</i> and <i>Salmonella enteritidis</i> in Shrimp Samples

A multiplex polymerase chain reaction (PCR) method was evaluated for simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enteritidis in shrimp samples. The sensitivity of DNA amplification by PCR in this method was found to be 103 cfu/ml for each pathogen. When this protocol was adopted for the detection of each of the above mentioned pathogen in spiked shrimp extract culture, similar sensitivity was observed. However, this method detected 1 bacterial cell for E. coli O157:H7 and S. enteritidis and 100 for L. monocytogenes per 25 g spiked shrimp samples after overnight enrichment. In the commercially imported shrimp samples, none was found to contain any of the three pathogens by multiplex PCR or by conventional method, which suggests that the multiplex PCR is a reliable and useful for rapid screening of shrimp samples for E. coli O157:H7, L. monocytogenes and S. enteritidis. This will save time and increase our ability to assure food safety. Keywords: Multiples PCR, Shrimp extract, Spiked sample, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enteritidisDOI: http://dx.doi.org/10.3329/bjm.v24i1.1236 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 42-46