High-throughput Suspension Array For Detecting Foodborne Pathogens

Food contaminated with pathogens can lead to various diseases and is a serious threat tohuman health. Pathogens that cause food-borne diseases are the most important factors affectingfood safety. Recently, numerous food poisoning cases have been caused by bacteria.Aside from the conventional techniques employed for detecting pathogens, newimmunologic methods and molecular biological methods have been described. Although theseapproaches have been successful for pathogen identification at the species and genotype levels,they are costly, time consuming, complicated, have poor sensitivity, or due to the amplificationprocess is not easy to control, likely to cause pollution false positive. Therefore, a rapid,accurate, sensitive multichannel method for detecting pathogens needs to be employed.Suspension array, an expeditious method for the simultaneous detection of several pathogens,was applied in the present study.We reported the development of a protocol to simultaneously detection of the pathogenswas constructed using a suspension array. This protocol reached the requirements of clinicaldetection limit.In first part, a new suspension array technology is proposed for the simultaneousquantitative determination of four pathogens in food. Four sets of primers and species-specificcapture probes were designed based on the16S rDNA gene sequences of Escherichia coli,Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella downloaded from GenBank.The specific nucleic acid probes were covalently bound to the surface of fluorescentmicrospheres for liquid suspension hybridization. The biotin-labeled PCR products obtainedfrom the samples were hybridized with their complementary nucleic acid probes, which weredetected after the addition of streptavidin phycoerythrin. No cross-reaction occurred among theproducts, and the sensitivity of the probe in the single-channel method was5×10-6mmol/L. Inthe multi-channel method, the sensitivities were as follows:5×10-4mmol/L for E. coli andSalmonella, and5×10-5mmol/L for S. aureus and V. parahaemolyticus. The multi-channelmethod achieved multidetection of several pathogens. For real sample detection, the methodsshowed a sensitivity of103CFU/g to100CFU/g of vegetables after enrichment for24h at37°C.This study demonstrates the utility of the suspension array specific for the16S rDNA gene fordetermining the presence of the four pathogens in food samples.In second part, multiplex polymerase chain reaction (PCR) was coupled to a suspension array to construct an assay for simultaneous identification of six kinds of pathogens. Thesuspension array allows the simultaneous detection of different target sequences in a multiplexand high-throughput format. The assay uses a liquid suspension hybridization format withspecific oligonucleotide probes covalently bound to the surface of fluorescent color-codedmicrospheres. Biotinylated target amplicons, hybridized to their complementary probesequences, are quantified by adding the conjugate, streptavidin R-phycoerythrin. Six probesderived from the sequence analysis of a specific gene were developed and validated. There waslittle cross-reaction among the various probes. In the multi-channel method, the detectionsensitivity was1.6×10-6mmol/L. Results of nucleic acid detection assays show detectionsensitivity for20to4×103CFU/ml, which is one to five orders of magnitude higher than thoseof PCR-agarose method in different bacteria. Aside from single channel capability, the assayallows the simultaneous detection of target genes in a single reaction, with detection limits of1-10CFU/ml. The accuracy, speed, flexibility, and sensitivity of the proposed assay arebeneficial for the diagnosis of pathogenic diseases.In third part, we prepared polyclonal antibodies of Shigella, Salmonella, and monocytesListeria, and antibodies labeled with biotin. Establish a quantitative detection of pathogenprotein suspension array; to optimize the experimental conditions; discussed the specificity,sensitivity, detection range, quantitative detection capabilities of the method and system tocompare with the commonly used in clinical double-antibody sandwich ELISA method. Thenewly developed suspension array appeared to be specific and sensitive, with the detectionsensitivities of about103CFU/ml,2.5×103CFU/ml,20ng/ml for Shigella, Salmonella, andmonocytes Listeria respectively, and in multiplex, the the detection sensitivities of about105CFU/ml,2.5×105CFU/ml,20ng/ml for Shigella, Salmonella, and monocytes Listeriarespectively, which were higher than those of corresponding conventional ELISA tests.Hence, a high-throughput technology has been adopted to identify pathogens. Data fromthe current study proved to be specific, sensitive, and flexible. The proposed method allowed acomplete array of different target species to be identified in a multiplex format. The assay can beexecuted in short time. Thus, the proposed assay is beneficial for applications in clinical settingsand epidemiological studies where there is a demand for a high-throughput system that allowsthe creation of multiple-testing platforms for routine testing. Further studies involving clinicalcultures and clinical specimens are under way.