Research On Visual And Efficient Detection Technique For Salmonella Based On Solid-phase Enrichment Assay And Nanobiosensor Technology

[Objective]Foodborne bacterial infections, especially infection by Salmonella, are serious problems for public health. Foodborne Diseases Active Surveillance Network (FoodNet) has an estimated 94 million cases of nontyphoidal Salmonella gastroenteritis and about 155,000 deaths. However, because of the insufficient sensitivity of the current detection and isolation techniques, the wild strains of this bacterium are not fully discovered. The real number of individuals with Salmonella in the public domain may be many times greater than the current reports. Since the minimum infectious dose of food-borne pathogens such as Salmonella is very low, there is urgent need to develop an appropriate technique to increase analytic sensitivity, detection and isolation rates. Though some one-step or rapid detection methods are emerging, the greatest demand is still the collection of bacterial isolates which is an important prerequisite for the etiological diagnosis, molecular epidemiological analysis and antibiotic sensitivity experiments. Actually, the successful detection and isolation highly depend on the sampling procedure* culture methods and the application of novel ultra-sensitive biotechnologies, i.e. biosensing technology, immunoassay technology and nanotechnology. Nowadays, it is a great challenge in nanotechnology for fluorescent nanobioprobes being applied to visually detect and directly isolate pathogens in situ. In this research, a novel immunosensor technique for efficient detection and visual isolation of Salmonella was designed and studied by applying fluorescent nanobioprobes on a specially designed cellulose-based swab (a novel solid-phase enrichment system). Furthermore, it is expected to develop a more simple and low-cost method based on the solid-phase enrichment system for Salmonella screening in many applications of large-sized samples related to public health surveillance.[Methods]1. Establishment of a solid-phase enrichment assay (or Salmonella detection.(1). Development of the swab and enrichment medium:Salmonella can produce hydrogen sulfide (H2S) in the course of growth and metabolism. According to the characteristic, a novel biochemical reaction system was designed to promote the generation of ferrous sulfide. The presence of Salmonella was observed by the development of ferrous sulfide (a black precipitate). As an empirical statistical technique, the central composite design (CCD) in design-Expert(?) software for response surface methodology (RSM) was used to optimize the composition of the chromogenic medium. Nine type Salmonella from different serogroups belonging to A-F and forty local Salmonella strains from different serotypes were divided into two groups as quadratic polynomial models.(2). Methodological evaluation:According to the Cumitechs (Cumulative Techniques and Procedures in Clinical Microbiology) from the American Society for Microbiology(ASM), mixed samples containing 101 cells per mL Salmonella typhimurium and 105 cells per mL, 106 cells per mL or 107 cells per mL E. Coli, were analyzed in groups using serial dilutions with purified water (40 samples in each group). The limit of detection (LOD), sensitivity, false-negative rate, specificity, false-positive rate were evaluated. In order to determine the difference of detection rate,668 fresh anal samples from health practitioners were collected from many Centers for Disease Control and Prevention (CDC) and tested by both the immunosensor technology and the traditional standard method.2. Visual and efficient detection technique for Salmonella based on solid-phase enrichment assay and nanobiosensor technology(1). Preparation of antibodies:Sixteen Japanese big ear rabbits(SPF level) were regularly immunized with whole cell antigen from eight type Salmonella belonging to different serogroups (A-F). Then their antibodies were extracted and purified on the stage of experiment and exploration.(2). Synthesis of probes:Using bifunctional crosslinkers, the conjugation of watersoluble amino-functionalized (NH2-PEG-NH2) CdSe/ZnS QDs with the antibodies was complied.(3). Development of optical observation instrument and detection procedure:All 156 LED lamp beads were made of a mixture of gallium nitride (GaN) and indium nitride (InN). This system provided adjustable excitation light (λex= 405 nm) and a total power of 10 watts (input voltage:90V-264V, output voltage:12V). The cut-off filter (Standard serial number: ZWB2) just covered the annular fluorescent light. Since the excited light and interferential light occurring in the wavelength range of 400-680 nm was blocked by the cut-off filter. The artificial LED light ring and cut-off filter were assembled and fixed on the outer layer of the objective lens of a stereomicroscope. With the application of bioprobe and instrument, the exploration of the detection and observation process was carried out.(4). Characteristics analysis of bioprobes:With transmission electron microscopy (TEM), the mAbs-conjugated QDs bioprobes attached onto the surface of Salmonella were recorded. The fluorescence absorption and emission spectra of these mAbs-conjugated QDs were recorded with an ultraviolet (UV) visible spectrophotometer and a fluorescence spectrophotometer. The surface conformation of Salmonella and mAb-conjugated QDs on the swab fiber were recorded with field emission scanning electron microscope (FESEM)(5). Methodological evaluation:Based on the Cumitechs from the ASM, with the mixed samples containing 101 cells per mL Salmonella and 105 cells per mL,106 cells per mL or 107 cells per mL non-Salmonella strains, including E. Coli, Proteus mirabilis or Citrobacter freundii, LOD was analyzed in groups using serial dilutions with purified water (40 samples in each group). The false-negative rate, false-positive rate and analytic was evaluated. In order to determine the sensitivity, specificity, detection rate and actual workload,120 fresh anal samples uncontaminated with Salmonella were collected by the CDC.101 cells per mL Salmonella typhimurium were added to the related test tubes of each sample for further evaluation with the newly developed assay and the traditional standard method.3. Visual detection technique for efficient screening and isolation of Salmonella based on solid-phase enrichment method using fiber membrane.(1). The novel solid-phase enrichment assay using fiber membrane, simple ultraviolet light-emitting diode and the visual detection technique were developed. And two chromogenic functionalized reactions on solid-phase membrane was developed. One reaction for screening is based on the sulfate-reducing capacity of Salmonella. Another is the Salmonella C8 esterase interacts with 4-methylumbelliferyl caprylate (MUCAP) to produce fluorescent umbelliferone (4-methylumbelliferone,4-MU), which is visible under ultraviolet light.(2). Methodological evaluation:With the 115 type and local strains, the special specificity of the visual detection technique was tested. And 1,304 anal samples from health practitioners were prepared by the the Center of Disease Control and Prevention (CDC). To observe the specificity and the effectiveness of methodology on Salmonella detection in anal samples from healthy people,101 cells mL-1 Salmonella typhi (ATCC 14028) were added to the related test tubes from 80 anal samples of uncontaminated Salmonella. To determine the practicality of the new detection technique,1,224 fresh anal samples were analyzed with the novel method and the traditional standard method.[Results]1. A solid-phase enrichment assay for Salmonella detection.(1). Development of the developed swab and enrichment medium:As a solid phase support (SPS), each swab contained 0.11 g of 100% medical-grade degreasing cotton with a 14.5 cm bamboo handle. The swabs were capable of absorbing approximately 600\iL medium or sample solution. An optimal medium formula for Salmonella was determined, which contained 40 μL of 10% polyvalent polypeptone,40 μL of 10% buffered peptone water,6 μL of 10% ferric citrate amine,6 μL of 20% sodium hyposulfite,6 μL of 5% cystine,6.4 μL of 20% sodium carbonate,8 μL of 1% sodium deoxycholate and μg of SSAP in 487.6 μL of distilled water. Values of "Prob> F" on the Model (Y1) and Model (Y2) less than 0.0001 and indicate the model terms for optimized medium formula were significant. (Chinese industry standards of medical absorbent cotton:YY/T 0330-2015)(2). Methodological evaluation:The LOD was 101 cells per mL Salmonella on the background of 105 cells per mL E. Coli. The sensitivity was 98.75% and false-negative rate was 1.25%. For the simulated samples containing 105 cells per mL E. Coli, the specificity was 100%and the false-positive rate was 0.0%. Through the detection and statistical analysis on 668 fresh anal samples, the positive rate was 2.10% with the novel technology and 0.45% with the traditional standard method.2. Visual and efficient detection technique for Salmonella based on solid-phase enrichment assay and nanobiosensor technology(1). Preparation of antibodies:All of antiserum titers were more than 1:2048, each concentration of purified antibodies were 1.38mg/mL,1.42mg/mL,1.58mg/mL, 1.07mg/mL,1.52mg/mL,0.78mg/mL,1.58mg/mL,1.34mg/mL respectively. On widal agglutination test, the agglutination titer of Salmonella typhi, Salmonella paratyphi A and B varied from 1:2048 to 1:4096.(2). Characteristics analysis of bioprobes:With gel electrophoresis of mAbs-conjugated QDs, the biological probes were successfully labeled. Under optimal conditions, the concentration of mAbs-conjugated QDs was 98.6×10-2 μg/μL. With TEM, the fluorescence emission spectras of water-soluble QDs before and after the conjugation were almost entirely consistent around 526nm. It was identified that the synthesis had little effect on the characteristics of QDs.(3). The visual fluorescent nanosensor technique and the optical observation device were successfully established. With FESEM, the immunological specificity of bioprobe was confirmed.(4). Methodological evaluation:The LOD was 101 cells per mL Salmonella on the background of 105 cells per mL non-Salmonella strains, including E. Coli, Proteus mirabilis or Citrobacter freundii. In the simulated samples containing 101 cells mL’1 Salmonella typhimurium, the detection rate was 96.67% with LSCM and 91.67% with a traditional culturebased method. The production rate of black spots was 100%(120/120), and the production rate of fluorescence was 68.33% with naked eyes. In the samples without Salmonella, the production rate of black spots was 25.00%(30/120). Correspondingly,75.00%(90/120) of samples could be directly judged as negative and excluded, and the production rate of fluorescence (false-positive rates) was 1.67% on the present method. The excluded samples (75.00%) in this study resulted in a significant decrease of subsequent workload including bacterial detection and isolation.3. Visual detection technique for efficient screening and isolation of Salmonella based on solid-phase enrichment method using fiber membrane.(1). Grade 3 chromatography papers was applied on the novel solid-phase enrichment assay. Every paper was exactly loaded with 1.0 mL of the developed medium. The SPS-based technique was developed based on a specially developed cultural method and two chromogenic reactions, which involved the production of H2S and a fluorescence test originated from 4-methylumbelliferyl caprylate reagent (MUCAP). (Chinese national standards of chemical analytical filter paper:GB/T 1914-2007)(2). Methodological evaluation:The species specificity was 94.78%(109 from 115) on the trial of 115 strains. And the LOD was 101 cells mL-1 Salmonella on the background of 105 cells mL-1 E. Coli. In samples containing Salmonella, the production rate of black spots or patches and the visible rate of fluorescence were 100%(80 from 80). In the trial of 1,224 fresh anal samples, the positive detection rate for Salmonella was 0.49%(6 from 1224) by the traditional method and 2.45%(30 from 1224) by our new method, the rate of H2S positive-MUCAP positive samples was 37.25%(456 from 1224), and the rate of H2S positive-MUCAP negative samples was 17.40%(213 from 1224). Using our new method, only the remaining H2S and MUCAP positive samples (465 from 1224,37.25%) were screened for further testing.[Conclusions]A specific, efficient, ultrasensitive and visual immunosensor technique and a novel, visual, simple and low-cost enrichment method using fiber membrane for Salmonella had both been established. They will be useful tools for screening and isolating in a large number of samples in the field of public health. Indeed, the efficient isolation of Salmonella will help to perform further molecular epidemiological analysis of public health surveillance (such as food poisoning and water pollution), etiological diagnosis and the drug sensitivity tests in clinic. It was believed that this new detection technique using chromatography membrane could be applied in the field of public health surveillance, especially be popular in developing countries.