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Developement and Applification of the Fluorescence-Labeled Bacteriophages for Detection of the Intestial Pathogens in Food

Author: JiangQin
Tutor: HuangKeHe
School: Nanjing Agricultural College
Course: Clinical Veterinary Medicine
Keywords: Salmonella Escherichia coli Shigella Fluorescence-labeled bacteriophages Detection
CLC: S854.43
Type: Master's thesis
Year: 2009
Downloads: 21
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Abstract

Salmonella, E.coli and Shigella are three of pathogenic enterobacteriaceae primarily involved in causing gastrointestinal illnesses, septicemia, and local imflammatory, and even with the symptoms of edema, dysenteria and nerve signs. Especially the 0157:H7, Sh. flexneri and Sh. dysenteriae exhibit pathogenicity strongly. The three intestinal pathogens are considered to be the causative agents up to 80% of all cases of diarrhea associated with the consumption of fishery products in the summer months. The raditional diagnostic tests for Salmonella, E. coli and Shigella are laborious and nucleic acid-based tests, and aways present flase negative results. Therefore, it was essential to develop a rapid, sensitive and specifical test for detection of the presence of these intestinal pathogens.The technique of fluorescent-labeled phages was relying on the characteristics of phages that were high specifically infected to their hosts, and injected the nucleic acid into. Fluorescence labeled the phages with the nucleic acids stain, the phages signal was amplified with the fluorescence microscope, and the pathogens should be detected specifically and efficiently. This method showed a high sensitivity, short period, visual and accuracy to intestinal pathogens detection. If the genera and species phages were labled with different nucleic acids stains, and matched with each other for detection pathogens, two more than pathogens should be detected simultaneously on a single filter membrane. The fluorescent-labeled phages matching offers rapid, sensitive, specific and simultaneous detection Salmonella, E. coli and Shigella, even the 0157:H7, Sh. flexneri and Sh. dysenteriae in food.In present paper, a new technique based on fluorescent-labeled phage for efficient detection of Salmonella and fluorescent-labeled matching phages for simultaneous and accurate detection of Salmonella, E. coli, Shigella and O157:H7, Sh. flexneri or Sh. dysenteriae were established. The genera specifical phages Salmonella-O-I, E. coli-E4, and Shigella-Sh were labled with SYBR(?) gold stain (an efficient nucleic acids dye), and the species specifical phages 0157:H7-LG1, Sh. flexneri-D2 and Sh. dysenteriae-2 were labeled with DAPI (another nucleic acids dye), respectively. Then a screenning of seafood in Eastern China for the presence of these three intestinal pathogens was carried out base on the fluorescent-labeled phages matching.1 Develop a rapid detection method for Salmonella in food by using specific Salmonella-phage O-I/SYBR(?) gold stainSalmonella is one of important human food pathogens. Traditional diagnostic tests for Salmonella are laborious and nucleic acid-based tests, and aways present flase negative results. Therefore, it is important to develop a rapid, sensitive and specifical test for detcetion of Salmonella. A specific Salmonella-phage O-I labeled with SYBR(?) gold stain was presented for fast detection of Salmonella in pure cultures and foods. The labeled phage O-I was high specificitify absorbed to the Salmonella, and injected its labeled nucleic acid into the host cell. With the fluorescence microscope amplified the phage signal, the Salmonella were detected. Treatment five kinds of bacteria from enterobacteriaceae with O-I/SYBR(?) gold stain, showed that all of the 40 Salmonella strains exhibited rod fluorescence, other bacteria including 10 Proteus,20 Shigella,20 E. coli and 10 Staphylococcus did not exhibit this feature. The sensitivity of detecting Salmonella was 8.5 CFU.100μL-1. The detection for 120 food samples by using the O-I/SYBR(?) gold stain had similar results to those by using the biochemical method. The accuracy rate was 91.7%, and it could be finished within 8 hours. Fluorescent-labeled O-I phage could rapidly, sensitively and specifically detect Salmonella species in food samples.2 Multiplex Fluorescent-labeled phages for simultaneous detection of Shigella, Sh. flexneri and Sh. dysenteriaeThree multiplex Fluorescent-labeled phages were used for the simultaneous and fast detection of Shigella, Sh. flexneri or Sh. dysenteriae on a single filter membrane. The specific Shigella-phage Sh labeled with SYBR(?) gold stain, Sh. flexneri-phage D2 and Sh. dysenteriae-phage labeled with DAPI, were tested on Shigella, Sh. flexneri and Sh. dysenteriae and other enterobacteriaceae bacteria species, each bacteria species infected by Sh and D2, or Sh and 2. With the different wavelength (510 nm～520 nm for SYBR(?) gold stain,450 nm～460 nm for DAPI) microscopic examination, Shigella, Sh. flexneri or Sh. dysenteriae could be detected simultaneous on a single filter membrane.The result showed that the phages were highly specific for Shigella, Sh. flexneri and Sh. dysenteriae respectively. The sensitivity of the phages was demonstrated to approximately 8.5 CFU.100μL-1. The detection for 120 food samples by using the Sh/SYBR(?) gold stain, D2/DAPI and 2/DAPI had similar results to those by using the biochemical method. The accuracy rate of Shigella was 95.7%, Sh.flexneri and Sh. dysenteriae were 100%.3 Developement of the fluorescence-labeled bacteriophages for simultaneous detection of three intestinal pathogens in foodThe Fluorescent-labeled bacteriophages matching was used for the simultaneous and fast detection of Salmonella, E.coli and Shigella, also detected the 0157:H7, Sh. flexneri or Sh. dysenteriae with E. coli or Shigella on a single filter membrane. The specific Salmonella, E.coli and Shigella-phage O-I, E4 and Sh were labeled with SYBR(?) gold stain, 0157:H7-phage LG1, Sh. flexneri-phage D2 and Sh. dysenteriae-phage 2 were labeled with DAPI. The matching phages were tested on Salmonella, E. coli and Shigella and other enterobacteriaceae bacteria species, each bacteria strain were infected by the genera and species labled phages matching, O-I, E4 and Sh/SYBR(?) gold stain labeled, and LG1, D2 and 2/DAPI labeled. With the different wavelength (510 nm～520 nm for SYBR(?) gold stain,450 nm～460 nm for DAPI) microscopic examination, the lytic patterns of phages were researched. Salmonella, E.coli and Shigella could be detected simultaneous. The result showed that the phages were specific for their hosts. The sensitivity of all the phages was demonstrated below to 10 CFU.100μL-1. The detection for 120 food samples by using the bacteriaphages matching had similar results to those by using the biochemical method. The accuracy rates of 0157:H7, Sh.flexneri or Sh. dysenteriae with E.coli and Shigella all were 100%. The matching phages could rapidly, sensitively, specifically and simultaneously detect Salmonella E.coli and Shigella species, also the 0157:H7, Sh. flexneri or Sh. dysenteriae in food samples.4 Screenning of Seafood in Eastern China for the Presence of Salmonella, E.coli and Shigella by fluorescence-labeled bacteriophages matchingTo understand the distibution of Salmonella, E.coli and Shigella, especially the 0157: H7, Sh. flexneri and Sh. dysenteriae in the coastal areas of eastern China, a total of 500 seafood samples were purchased from several local markets in harvest season in eastern China. The presence of Salmonella, E.coli, Shigella,0157:H7, Sh. flexneri or Sh. dysenteriae was determined by fluorescence-labeled bacteriophages matching and VITEK 2 identification system based on culture method. As a result of bacteriophages matching, the positive ratio of Salmonella was 16.6%, and E.coli and Shigella was 9.6%,7.4%, repectively. The proportion of 0157:H7 to E.coli was 52.1%, Sh. flexneri to Shigella was 29.7% and Sh. dysenteriae was 35.1%, repectively. The apparently high number of Salmonella,O157:H7, Sh.flexneri and Sh. dysenteriae were detected by the bacteriophages matching method in the substrates examined. It is concluded that retail seafood was commonly contaminated with the three main enterobacteriaceae pathogens, especially the 0157:H7, Sh. flexneri and Sh. dysenteriae and could serve as a potential risk for the consumers in eastern China if proper hygienic and cooking considerations are a miss.

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