Development And Application Of High Throughput Pathogen Detection System Based On Multiplex Real-time PCR And Suspension Array

Multiplex real-time PCR and suspension array have recently been demonstrated to be rapid, sensitive, specific and high throughput assay for detection of pathogens in clinical and food safety. In this study, key technology and whole solving scheme for virus detection by multiplex real-time RT-PCR and food-borne pathogens identification by suspension array were investigated and provided.Total virus nucleic acid extraction mediated by paramagnetic particles was developed. The method described here excelled other reagents in nucleic acid outcome and purity and could be automated by robot. The kit contained: Lysis buffer (4.5mol/L guanidin thiocyanate,50 mmol/L Tris-HCl,10 % Triton-100,20 mmol/L EDTA,pH 6.0), concentrated elution buffer 1 (4.5mol/L GuanidinThiocyanate,46 mmol/L Tris-HCl,pH 6.0), 40% (V/V) ethanol should be added before use; elution buffer 2 (75% ethanol); elution buffer 3 (ethanol); magnetic beads (Promega).Universal multiplex PCR buffer system for DNA and RNA templates were established respectively by successfully overcoming frequent faced obstacles in multiplex PCR system. DNA templates buffer contained: 60mmol/L Tris-HCl (pH 8.3), 15mmol/L KCl, 8mmol/L ( NH4) 2SO4, 4mmol/L MgCl2, 1.25mmol/L dNTP,0.5μL DMSO. RNA templates buffer contained: 60mmol/L Tris-HCl (pH 8.3),10mmol/L KCl, 16mmol/L ( NH4) 2SO4, 5mmol/L MgCl2, 1. 5mmol/L dNTP,1.0mmol/L Betaine,0.5μL DMSO,0.5μL 1mg/mL gp32. Identical volume of anti-Taq to Taq should be added to the buffer before PCR start.The method for producing of armored virus with His tag, which acted as positive control or internal control, was described, thus provided a new internal control model throughout the detection. Universal expressing plasmid was successfully constructed by inserting 6 histidines toβ-ring of MS2 bacteriophage coat protein with the help of molecular manipulation. Armored virus contained an exogenous RNA sequence in high concentration and super purity was simply obtained by induction and Ni+ chromatography purification. The Armored virus can be conserved in SM buffer for more than one year at 4℃or -20℃.Assays to detect highly virulent Chinese-type isolate of Porcine Reproductive and Respiratory Syndrome viruses, acute hemorrhagic conjunctivitis agents and HFMD viruses were established by multiplex real-time RT-PCR respectively. The specificities for all assays were 100% in the selected panels. The sensitivity of all the assays were evaluated and excelled those methods established before. Intra- and inter-assay reproducibility of the developed assays was all under 2.2% which indicated the repeatability and confirmed their sensitivity. All the developed assays demonstrated their applicability when hundreds of suspicious clinical specimens were analyzed and identical to the results of normal PCR pulsing sequencing or virus isolation followed by neutralization tests simultaneously. The occurrence of PCR inhibition with stools, rectal swabs and throat swabs, from1.8% to3.4%, confirmed that application of internal controls to monitor sample extraction and amplification inhibitors was necessary to prevent false negatives.A selective enrichment broth was formulated originally for simultaneous growth of Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogens. The medium contained: 17 g of tryptone, 3 g of peptone, 15 g of sodium chloride, 2.5 g of disodium hydrogen phosphate, 2.5 g of glucose, 10mg Nalidixic acid, 2g lithium chloride, and 1mg potassium tellurite, 2.5g sodium pyruvate and 5g mannitol per liter distilled water. The broth inhibited the growth of non-target organisms well while supported the growth of target bacteria in similar growth speed and could yield cell densities of more than 107 CFU/mL after 24 h incubation at 37℃.A selective enrichment broth was formulated firstly for simultaneous growth of Salmonella, Vibrio parahemolyticus, and Vibrio cholerae. The enrichment broth was obtained by supplementing the BPW with 2.5g bile salt no. 3, 1mg potassium tellurite and 20g sodium chloride as inhibitors,and 1.25g glucose, 1.25g mannitol, 1.25g hydrous sodium sulfite and 5 mg sodium pyruvate as accelerants. The broth inhibited the growth of non-target organisms well while supported the growth of target bacteria in similar growth speed and could yield cell densities of more than 105 CFU/mL after 18 h incubation at 37℃.Detection method of the frequent found food-borne pathogens in meat and seafood by suspension array was originally developed. The specificity was 100% in the selected panel and the sensitivity was evaluated to be 100 CFU/mL and equal to the result of real-time PCR. Intra- and inter-assay reproducibility of the developed assay was under 2 % which indicated the repeatability and confirmed its sensitivity. Three important factors in affecting the detection signal were investigated and summarized: 1) Amplicons for target bacteria should be around 100bp. 2) Detection signal might be added by more than 65% if the upper primers'5'terminal was also marked with biotin. 3) It was important to select an appropriate temperature for anneal between the probes and the amplicons.