Study On Detection Methods For Yersinia Enterocolitica In Human Feces

BackgroundsInfectious diarrhea which is caused by a wide variety of pathogenic microorganisms and their metabolites or parasites does great harm to human health, and also has a severe impact on public health, bringing a series of social and economic problems. Rapid, sensitive and specific detection and diagnosis for pathogens that lead to diarrheal disease in time are of great significance to the prevention and control of outbreaks and prevalence of infectious diarrhea.Yersinia enterocolitica, an important pathogen of infectious diarrhea, has been of great concern worldwide in recent years and intake of contaminated food may be the leading transmission route contributing to human infection. Infection with Yersinia enterocolitica in humans causes various diseases, and the most common one is acute gastroenteritis with the main clinical manifestations of fever, abdominal pain and diarrhea. Besides, it may be the causal agent of extraintestinal symptoms including erythema nodosum, reactive arthritis, granulomatous hepatitis, meningitis, myocarditis, iritis, deep abscess, septicemia, wound infection and sometimes even contribute to autoimmune disorder like Graves' disease via superantigen. Routine detection of suspected individuals for Yersinia enterocolitica infection is based upon bacteria isolation, and subsequent biochemical test identification, as well as PCR assay and Real-time PCR assay. The conventional isolation method has been considered as the gold standard for diagnosis, but the whole identification process is laborious, time consuming and less sensitive. PCR-based molecular biological methods (PCR or real-time PCR) require expensive instruments, high detection cost and skilled personnel, which may delay their application in field rapid detection and in peripheral health care settings and clinics. A rapid and simple new detection method for Yersinia enterocolitica is significantly required, and has become the hot spot of research.Loop mediated isothermal amplification (LAMP) is a sensitive, specific and convenient strand- displacement gene amplification technique newly emerging as a simple and rapid diagnostic tool for early detection and identification of microbial diseases in recent years.The whole procedure is very simple and rapid wherein the amplification employs a set of specifically designed primers of a target gene and can be completed by incubating all the reagents in a single tube under isothermal conditions. The LAMP method has successfully detected many kinds of pathogenic organisms, including bacteria, viruses, fungi and parasites. Considering its advantages of isothermal amplification, simple operation, high amplification efficiency, easy detection by agarose gel electrophoresis or visual observation together with high sensitivity and specificity, LAMP has potential applications for pathogen detection especially in field rapid detection, open country detection during wartime and peripheral health care settings and clinics.Recent studies on the detection of Yersinia enterocolitica by LAMP have focused merely on contaminated foods, though great sensitivity and specificity were achieved. Whether the method can be applied in feces and what the detection results are have not been reported in China or in other countries until now.The LAMP reaction was traditionally optimized by the comprehensive-test method or one-factor-at-a-time method by a majority of researchers. These methods usually involve a relatively large number of experiments which are labour intensive, and time-consuming, but they may not be able to completely guarantee the determination of the optimal conditions. In addition, there have not been any reports on quantifying analysis of the main factors influencing the LAMP reaction.Systematic review is an important method of Evidence-Based Medicine, in which qualitational and quantificational analyses are conducted on a specific clinical or research issue in the purpose of acquiring the best evidence for guidance. Thousands of papers about systematic review involving all areas of medical research have been published, including etiopathogenisis, diagnostic tests, prevention and treatment evaluation, prognosis and so on. So far, there have been many reports on Yersinia enterocolitica detection by nucleic acid amplification including PCR, Real-time PCR. Nevertheless, no systematic review has been found to evaluate these methods.ObjectivesThe present study aimed to:1. Synthetically appraise the studies using systematic review on Yersinia enterocolitica detection by nucleic acid amplification(PCR, Real-time PCR and LAMP) which have already been reported at present;2. develop a LAMP method for detecting Yersinia enterocolitica in human feces, optimize LAMP reaction condition based on orthogonal design and then further analyze the main factors that would influence LAMP reaction;3. determine the specificity and sensitivity of the optimized LAMP method, which was also applied in clinical faecal samples. The conventional isolation method, PCR assay and Real-time PCR assay were conducted parallelly and compared with the developed LAMP method to evaluate its application.Methods1. MEDLINE, EMBASE, BIOSIS, Web of Science, Cochrane Library, WANFANG DATA, VIP INFORMATION, CNKI (Jan.1st,1990-Dec.31st,2010) were searched on studies of the main nucleic acid amplification technology for the detection of Yersinia enterocolitica. Quality of included literatures was assessed by QUADAS (quality assessment of diagnostic accuracy studies). The software Meta Disc1.4 was used to summarize the data.2. LAMP primers directing ail gene specific for pathogenic Yersinia enterocolitica were designed using the online software Primer Explorer version 4. According to the orthogonal table L16(45),16 sets of treatment repeated twice, a total of 32 experiments were investigated, and the optimal amplification reaction system was determined in the employment of direct comparison, range analysis and variance analysis. The main factors that affect the LAMP amplification were analyzed simultaneously;3. Blast of NCBI was utilized to test the specificity of LAMP primers including the outer primers and inner primers.3 strains of Yersinia enterocolitica, and 18 non-Yersinia other bacterial strains were also examined to further assure specificity of the established LAMP method. Sensitivity of the LAMP method was evaluated in Yersinia enterocolitica genome DNA, pure culture bacteria and artificially infected faecal specimens, with PCR and Real-time PCR as parallel detection at the same time. In addition,206 faecal specimens from diarrheal patients and 50 normal specimens were detected for pathogenic Yersinia enterocolitica by culture, PCR, Real-time PCR and LAMP in order to make a comparison among them. Results1.12 relevant studies from 11 literatures were selected according to the standards. Data synthesis was performed using random effect model. Pooled accuracy indicators of DOR(diagnostic odds ratio), sensitivity, specificity, positive LR (likelihood ratio) and negative LR were 458.91 (95%CI:72.62-2900.09),0.72 (95%CI:0.69-0.75),0.96 (95%CI:0.95-0.96),27.23(95%CI:10.60-69.96) and 0.09(95%CI:0.02-0.30) respectively. High heterogeneity was found and all these summary measures of diagnostic accuracy were not clinically meaningful(P<0.1, I2>50%), but there was no threshold effect.2. Four LAMP primers were designed corresponding to the ail gene specific for pathogenic Yersinia enterocolitica. The optimal LAMP condition was containing 0.8μM each inner primer(FIP and BIP),0.2μM each outer primer(F3 and B3), 1.0mM dNTPs,4.0mM MgCl2,1.0M Betaine,8U of Bst DNA polymerase, 2.5μl of 10×Thermopol buffer and 5μl DNA template in 25μl volumes, which was confirmed by direct comparison, range analysis and variance analysis after 32 LAMP reactions were completed according to the orthogonal table L16(45). And the amplification program was as follows:60℃for 60 min and then heated at 80℃for 4min. The optimized LAMP reaction system was proved to be repeatable, reliable and stable. Results from analysis of variance displayed that the order of amplification effect of the five main factors were FIP/BIP, dNTPs, Mg2+, F3/B3, Betaine in turn under conditions of the present study.3. (1)Both the designed inner primer FIP/BIP and outer primer B3/F3 were found to have 100% homology with Yersinia enterocolitica by Blast of NCBI. Only genomic DNA from Yersinia enterocolitica strains was positively detected by LAMP, while no LAMP products were obtained when detecting non-Yersinia strains, which showed the good specificity of the method. (2)When the reference strain was used as the test organism, the LAMP method can detect Yersinia enterocolitica genomic DNA as low as 5.3fg (observed by gel electrophoresis or visual detection under UV light) or 53fg (visual detection under normal light)/25μl reaction system minimally; When detecting template DNA from pure culture bacteria, the LAMP method was capable of detecting 1.5 CFU/ml (observed by gel electrophoresis or visual detection under UV light) or 15 CFU/ml (visual detection under normal light) at the least; in artificially infected faecal specimens, the detection limit of the LAMP method was 15 CFU/g (observed by gel electrophoresis or visual detection under UV light) or 150 CFU/g (visual detection under normal light). In comparison, it was possible for PCR to detect the target sequence with the same detection limit as LAMP investigated by visual inspection under normal light. However, PCR was found to be less sensitive than LAMP observed by gel electrophoresis or visual detection under UV light. Real-time PCR had the same detection limit as LAMP observed by gel electrophoresis or visual detection under UV light. The culture method can isolate the bacteria at concentrations above 1.5×103CFU/g from feces. Thus, LAMP here we introduced was 10 times more sensitive than PCR, had the same detection limit as Real-time PCR, and was 100 times more sensitive compared to culture method.4. (3) With regard to the 206 clinical specimens from diarrhea patients,11 were tested positive using the LAMP assay by visual detection or agarose gel electrophoresis, which were in accordance with PCR and Real-time PCR. But only 9 of them were isolated and identified successfully by the culture method. All the four methods failed to detect pathogenic Yersinia enterocolitica in 50 normal feces from healthy individuals. The specificity of LAMP was calculated to be 99.0% with culture as the "gold standard", all of the nine culture-positive specimens were also positive for LAMP and the Youden index was 0.990. The two methods showed no significant difference (P=0.500) and had great agreement with each other (Kappa=0.895, P=0.000)Conclusions1. Generally, PCR, Real-time PCR and the LAMP assay for detection of Yersinia enterocolitica had a high test performance, but there was great heterogeneity among each study. Also, these accuracy measures like sensitivity (9%-100%) and specificity (37%-100%) had an extremely wide range. Therefore, these methods can not be used alone for Yersinia enterocolitica detection in place of traditional culture method, but the application of them combined with the culture method was recommended. And large epidemiological surveys were still required to assess these methods.2. A LAMP method was successfully developed to detect the pathogenic Yersinia enterocolitica directing ail gene in the present study. The optimized LAMP method was proved to be sensitive and specific, and can be completed in one hour using thermostatic water bath without elaborate and expensive instruments and special reagents. Also, the amplification results can be observed by several endpoint detection methods, but there was some difference in the detection limits.3. The established LAMP method was suitable for Yersinia enterocolitica detection in faecal samples. The detection process in feces comprised the cold enrichment overnight, DNA extraction together with LAMP amplification and result identification, which took less than 1 day to be accomplished. The new method showed a satisfactory concordance with the conventional culture method, PCR and real-time PCR method when being applied in clinical specimens.4. Orthogonal design combined with variance analysis was easy to operate and time-saving in the optimization of LAMP reaction system compared with comprehensive-test method or single-factor method and was more accurate and reliable than the simple analytical methods of direct comparison and range analysis. So the novel method was a considerable method to optimize LAMP reaction system.5. FIP/BIP, F3/B3, dNTPs, Mg2+ and Betaine concentrations all played important roles in LAMP amplification, and the order of effects of those factors was FIP/ BIP, dNTPs, Mg2+, F3/B3 and Betaine.