En høj overførselshastighed Platform for Screening af Salmonella spp. /Shigella spp.
Summary
Salmonella spp. /Shigella spp. er fælles patogener tilskrives diarré. Her, vi beskriver en høj overførselshastighed platform for screening af Salmonella spp. /Shigella spp. ved hjælp af real-time PCR kombineret med guidet kultur.
Abstract
Fækal-oral transmission af akut gastroenteritis forekommer fra tid til anden, især når folk, der håndteres fødevarer og vand er smittet med Salmonella spp./Shigella spp. Guld standard metoden til påvisning af Salmonella spp./Shigella spp. er direkte kultur, men dette er arbejdskrævende og tidskrævende. Her beskriver vi en høj overførselshastighed platform for Salmonella spp./Shigella spp. screening, ved hjælp af real-time polymerase kædereaktion (PCR) kombineret med guidet kultur. Der er to større faser: real-time PCR og guidet kultur. For den første fase (real-time PCR) vi forklare hvert trin i metoden: prøve samling, før berigelse, DNA-ekstraktion og real-time PCR. Hvis real-time PCR er positiv, så den anden fase (guidede kultur) udføres: selektiv kultur, biokemiske identifikation og serologiske karakterisering. Vi også illustrere repræsentative resultater, der genereres fra det. Protokollen beskrevet her ville være en værdifuld platform for hurtig, specifikke, følsomme og høj overførselshastighed screening af Salmonella spp. /Shigella spp.
Introduction
Diarré er stadig en fælles spørgsmål om sundhed med en høj incidens Vurder globalt1,2. Selvom dødeligheden er relativt lav, nogle patienter viser forskellige symptomer for uger (f.eks. løs og vandig afføring, en hastesag at gå på toilettet), hvilket gør den socioøkonomiske indvirkning meget høj3,4. Mere alvorligt, nogle patienter kan selv udvikle irritabel tyktarm, hvis venstre ubehandlet5. Der findes mange forskellige slags bakterier, virus og parasitter, der kan forårsage diarré6. Salmonella spp. /Shigella spp. er blandt de mest almindelige bakterier til transmission af akut gastroenteritis7,8,9,10,11. Derfor, mange amter har udstedt love eller regler for regelmæssig Salmonella spp. /Shigella spp. screening blandt mennesker, der vil håndtere mad og vand. For eksempel, den kinesiske regering har udstedt love for obligatorisk Salmonella spp. /Shigella spp. screening en gang om året.
Metoden guldstandarden for Salmonella spp. /Shigella spp. opdagelse er bakterier kultur. Gennem bakterier kultur og successive biokemiske identifikation og serologiske karakterisering, kan vi identificere arterne af bakterier, som kan lette sygdom udbrud management og antimikrobiel profilering for at støtte til behandling af patienter 12. det kan også hjælpe med at spore kilden til infektionen i løbet af Salmonella spp. /Shigella spp. udbrud13. Men denne metode er arbejdskrævende (kræver manuel betjening) og tidskrævende (tage flere dage), især for test af et stort antal prøver7. Derudover levedygtige men ikke-bestemmelse (VBNC) Salmonella spp. /Shigella spp.kan findes i nogle afføringsprøver14. I betragtning af disse ulemper, mange laboratorier har forsøgt at udvikle nye teknikker til påvisning af Salmonella spp. /Shigella spp.15,16,17,18 , 19 , 20 , 21 , 22 , 23 , 24 , 25. alle disse metoder bruger nukleare syre forstærkning test (NAAT), blandt hvilke Polymerasekædereaktionen (PCR) er den mest almindelige. En stor begrænsning af disse NAAT baseret metoder er, at døde bakterier, selv bakteriel snavs indeholder ufuldstændige genomisk DNA, kunne vise positive resultater26, som i vid udstrækning kunne påvirke de præcis diagnose af sygdommen. Blanco et al. viste, at Molekylær analyse er meget følsomme, ikke kun til levedygtige Salmonella i kulturer, men også til delvis genomer og døde eller ikke-levedygtige bakterier fra tidligere infektioner eller kontaminering26. Derfor bør der udvikles ny teknologi.
Her, beskrev vi en roman metode at kombinerer NAAT baseret metode og dyrkning. Som vist i figur 1, denne nye metode gælder real-time PCR screening først og derefter positive prøver sendes til bakterier kultur og identifikation.
Protocol
Representative Results
Discussion
Siden Salmonella spp. /Shigella spp. er ofte forbundet med madforgiftning og fækal-oral transmission af akut gastroenteritis28,29 og rutinemetoden er enten arbejdskrævende eller tidskrævende 7, vi beskriver en høj overførselshastighed platform for Salmonella spp. /Shigella spp. screening, ved hjælp af real-time PCR kombineret med guidet kultur.
Der er flere trin, der har…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af videnskab og teknologi Program af Zhuhai, Kina (grant nummer 20171009E030064), videnskab og teknologi Program af Guangdong, Kina (grant nummer 2015A020211004) og videnskab og teknologi Program af General Administration for kvalitetsovervågning, inspektion og karantæne i Folkerepublikken Kina (grant nummer 2016IK302, 2017IK224).
Materials
| Tris | Sigma | 10708976001 | |
| EDTA | Sigma | 798681 | |
| NP40 | Sigma | 11332473001 | |
| ddH2O | Takara | 9012 | |
| PrimeSTAR HS (Premix) | Takara | R040Q | |
| Nutrient Broth | LandBridge | CM106 | |
| Nutrient agar | LandBridge | CM107 | |
| Selenite Cystine medium | LandBridge | CM225 | |
| XLD | LandBridge | CM219 | |
| MAC | LandBridge | CM908 | |
| Salmonella chromogenic agar | CHROMagar | SA130 | |
| Salmonella diagnostic serum | Tianrun | SAL60 | |
| Shigella diagnostic serum | Tianrun | SHI54 | |
| anal swab (collecting tube plus) | Huachenyang | ||
| slide | Mingsheng | 7102 | |
| micro-loop | Weierkang | W511 | |
| incubator | Jinghong | DNP-9082 | |
| autoclave | AUL | SS-325 | |
| dry bath | Jinghong | KB-20 | |
| automated microbial identification system | bioMérieux | VITEK2 | other equivalent system could be used |
| fluorescent real-time PCR machine | ThermoFisher | ABI7500 | other equivalent machine could be used |
Riferimenti
- Roy, S. L., Scallan, E., Beach, M. J. The rate of acute gastrointestinal illness in developed countries. Journal of Water and Health. 4, 31-69 (2006).
- Wilking, H., et al. Acute gastrointestinal illness in adults in Germany: a population-based telephone survey. Epidemiology and Infection. 141 (11), 2365-2375 (2013).
- Friesema, I. H. M., Lugnér, A. K., van Duynhoven, Y. T. H. P. Costs of gastroenteritis in the Netherlands, with special attention for severe cases. European Journal of Clinical Microbiology & Infectious Diseases. 31 (8), 1895-1900 (2012).
- Henson, S. J., et al. Estimation of the costs of acute gastrointestinal illness in British Columbia, Canada. International Journal of Food Microbiology. 127 (1-2), 43-52 (2008).
- Okhuysen, P. C., Jiang, Z. D., Carlin, L., Forbes, C., DuPont, H. L. Post-diarrhea chronic intestinal symptoms and irritable bowel syndrome in North American travelers to Mexico. The American Journal of Gastroenterology. 99 (9), 1774-1778 (2004).
- Wongboot, W., Okada, K., Chantaroj, S., Kamjumphol, W., Hamada, S. Simultaneous detection and quantification of 19 diarrhea-related pathogens with a quantitative real-time PCR panel assay. Journal of Microbiological Methods. 151, 76-82 (2018).
- Van Lint, P., De Witte, E., Ursi, J. P., Van Herendael, B., Van Schaeren, J. A screening algorithm for diagnosing bacterial gastroenteritis by real-time PCR in combination with guided culture. Diagnostic Microbiology and Infectious Disease. 85 (2), 255-259 (2016).
- Liu, J., et al. Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study. Lancet. 388 (10051), 1291-1301 (2016).
- Wang, S. M., et al. Surveillance of shigellosis by real-time PCR suggests underestimation of shigellosis prevalence by culture-based methods in a population of rural China. Journal of Infection. 61 (6), 471-475 (2010).
- Wikswo, M. E., Hall, A. J. Outbreaks of acute gastroenteritis transmitted by person-to-person contact–United States, 2009-2010. MMWR Surveillance Summaries. 61 (9), 1-12 (2012).
- Shen, H., et al. The 12 Gastrointestinal Pathogens Spectrum of Acute Infectious Diarrhea in a Sentinel Hospital, Shenzhen, China. Frontiers in Microbiology. 7, 1926 (2016).
- Tariq, A., et al. Molecular profiling of antimicrobial resistance and integron association of multidrug-resistant clinical isolates of Shigella species from Faisalabad, Pakistan. Canadian Journal of Microbiology. 58 (9), 1047-1054 (2012).
- Ferrari, R. G., Panzenhagen, P. H. N., Conte-Junior, C. A. Phenotypic and Genotypic Eligible Methods for Salmonella Typhimurium Source Tracking. Frontiers in Microbiology. 8, 2587 (2017).
- Oliver, J. D. The viable but nonculturable state in bacteria. The Journal of Microbiology. 43, 93-100 (2005).
- Rintala, A., Munukka, E., Weintraub, A., Ullberg, M., Eerola, E. Evaluation of a multiplex real-time PCR kit Amplidiag(R) Bacterial GE in the detection of bacterial pathogens from stool samples. Journal of Microbiological Methods. 128, 61-65 (2016).
- Wohlwend, N., Tiermann, S., Risch, L., Risch, M., Bodmer, T. Evaluation of a Multiplex Real-Time PCR Assay for Detecting Major Bacterial Enteric Pathogens in Fecal Specimens: Intestinal Inflammation and Bacterial Load Are Correlated in Campylobacter Infections. Journal of Clinical Microbiology. 54 (9), 2262-2266 (2016).
- Van Lint, P., et al. Evaluation of a real-time multiplex PCR for the simultaneous detection of Campylobacter jejuni, Salmonella spp., Shigella spp./EIEC, and Yersinia enterocolitica in fecal samples. Eur Journal of Clinical Microbiology Infect Dis. 34 (3), 535-542 (2015).
- Kamkamidze, G., et al. Rapid Identification Of The Etiological Factors Causing Diarrheal Diseases. Georgian Medical News. (258), 89-92 (2016).
- Li, Y. Establishment and Application of a Visual DNA Microarray for the Detection of Food-borne Pathogens. Analytical Sciences. 32 (2), 215-218 (2016).
- Zhuang, L., et al. Detection of Salmonella spp. by a loop-mediated isothermal amplification (LAMP) method targeting bcfD gene. Letters in Applied Microbiology. 59 (6), 658-664 (2014).
- Shi, X. L., et al. Rapid simultaneous detection of Salmonella and Shigella using modified molecular beacons and real-time PCR. Zhonghua Liu Xing Bing Xue Za Zhi. 27 (12), 1053-1056 (2006).
- Mo, Q. H., et al. Preparation of a 96-microwell plate DNA diagnostic chip for detection of foodborne bacteria and its application in an incident of food poisoning. Nan Fang Yi Ke Da Xue Xue Bao. 30 (3), 417-421 (2010).
- Wang, H. B., et al. Probe-free and sensitive detection of diarrhea-causing pathogens using RT-PCR combined high resolution melting analysis. Biologicals. 44 (5), 360-366 (2016).
- Sun, H., et al. Rapid simultaneous screening of seven clinically important enteric pathogens using a magnetic bead based DNA microarray. World Journal of Microbiology and Biotechnology. 27 (1), 163-169 (2011).
- Qi, W., et al. Multiplex PCR assay for rapid detection of five important pathogenic vibrios. Chinese Journal of health laboratory technology. (24), 3497-3500 (2014).
- Blanco, G., Diaz de Tuesta, J. A. Culture- and molecular-based detection of swine-adapted Salmonella shed by avian scavengers. Science of the Total Environment. 634, 1513-1518 (2018).
- Tang, X. J., Yang, Z., Chen, X. B., Tian, W. F., Tu, C. N., Wang, H. B. Verification and large scale clinical evaluation of a national standard protocol for Salmonella.spp./Shigella.spp. screening using real-time PCR combined with guided culture. Journal of Microbiological Methods. 145, 14-19 (2018).
- Dekker, D. M., et al. Drinking water from dug wells in rural ghana–salmonella contamination, environmental factors, and genotypes. International Journal of Environmental Research and Public Health. 12 (4), 3535-3546 (2015).
- Gargano, J. W., et al. Mortality from selected diseases that can be transmitted by water – United States, 2003-2009. Journal of Water and Health. 15 (3), 438-450 (2017).
- Kumar, R., Surendran, P. K., Thampuran, N. Evaluation of culture, ELISA and PCR assays for the detection of Salmonella in seafood. Letters in Applied Microbiology. 46 (2), 221-226 (2008).
- Herrera-Leon, S., et al. Blind comparison of traditional serotyping with three multiplex PCRs for the identification of Salmonella serotypes. Research in Microbiology. 158 (2), 122-127 (2007).
- Cunningham, S. A., et al. Three-hour molecular detection of Campylobacter, Salmonella, Yersinia, and Shigella species in feces with accuracy as high as that of culture. Journal of Clinical Microbiology. 48 (8), 2929-2933 (2010).
- Eriksson, E., Aspan, A. Comparison of culture, ELISA and PCR techniques for salmonella detection in faecal samples for cattle, pig and poultry. BMC Veterinary Research. 3, 21 (2007).
- Dutta, S., et al. Sensitivity and performance characteristics of a direct PCR with stool samples in comparison to conventional techniques for diagnosis of Shigella and enteroinvasive Escherichia coli infection in children with acute diarrhoea in Calcutta, India. Journal of Medical Microbiology. 50 (8), 667-674 (2001).
