| BackgroundsYersinia enterocolitica, an important pathogen of infectious diarrhea, has been of great concern worldwide since the1980s, and may be the leading transmission route contributing to human infection. When it was found in New York in1933, it has caused several large outbreak of extragastrointestinal tract and gastrointestinal infection. In the mid-80s of last century, in China, it has caused twice epidemic, resulting in500people infected.Yersinia enterocolitica is a global distribution of food-borne pathogens that can cause various intestinal symptoms which are self-limiting disease., such as diarrhea, enteritis, mesenteric lymphadenitis and terminal ileitis. What make the researchers more concern is that it can cause a series of extraintestinal symptoms by spreading through the lymphatic system, such as reactive arthritis, erythema nodosum, endocarditis, and even sepsis, resulting in death. In recent, Yersinia enterocolitica is not yet included in the routine test items in the domestic hospital. In general, medical personnel are lack of understanding of the Yersinia enterocolitica and clinicians rarely consider Yersinia enterocolitica infection in the diagnosis of gastrointestinal tract. People may underestimate the real situation of the infection of Yersinia enterocolitica.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 including cold enrichment, selective enrichment biochemical and more than a dozen of biochemical identification, is time-consuming, less sensitive, laborious and subjective. The low quantity of Yersinia enterocolitica infection and the slow proliferation cause the low success rate of separation, and may also delay the use of antibiotic with Yersinia enterocolitica infection in patients. 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 the field of 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 which can amplify109-1010copies of the target gene with the use of Bst DNA polymerase, two pairs of specifically designed primers from six regions in a target gene and dNTPs, efficaciously, quickly, within1h under isothermal conditions.With and Also the products can be detected by visual observation. With its low cost, high amplification efficiency, high sensitivity, specificity efficiency and high practicability, it was gradually applied in various fields of life sciences, such as:pathogens and their resistance diagnostics, gene diagnosis and treatment, embryo sex identification, food hygiene inspection and environmental monitoring. LAMP has potential applications for pathogen detection especially in the field of rapid detection, open country detection during wartime and peripheral health care settings and clinics.In general, the LAMP reaction was 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 laborious, and time-consuming, blindness, so they may not be able to completely guarantee the determination of the optimal conditions. It has been reported that the LAMP reaction was optimized by orthogonal design which can reduce a number of tests. But we must know that part or most of the interaction of factors in this method would lose, and the orthogonal design is not easy.Factorial design (full factorial experimental design) is a comprehensive and balanced method which is composed by each of factors in different levels, and all combinations will repeat at least2independent experiments. The advantage of factorial design is that it can not only obtain a great of information so as to estimate the main effects of the experimental factors, but also the interaction effects at all experimental factors. Since the factorial design was founded in the1930s by famous British statistician RA Fisher, and was applied in agricultural experiment for the first time, factorial design has been a full development and wide application in industry, medicine, and science experiment so far.Objectives1. Develop a LAMP method for detecting Yersinia enterocolitica in human feces, optimize LAMP reaction condition based on factorial design and then further analyze the main factors that would influence LAMP reaction;2. Determine the specificity and sensitivity of the optimized LAMP method, which was also applied in clinical faecal samples. PCR assay was conducted parallelly and compared with the developed LAMP method to evaluate its application.Methods1. Choose LAMP primers from literature. Select FIP/BIP, MgCl2, dNTPs as the main factors affecting the LAMP reaction to establish factorial design table of4X4×4. According to the design table,64sets of treatment repeated twice, a total of128treatments were investigated and the product of LAMP was determinate by OD400-The optimal amplification reaction system was determined in the employment of variance analysis and multiple comparisons with the use of SPSS16.0and the main factors that affect the LAMP amplification were analyzed simultaneously;2. Extract the DNA from different samples in the employment of the bacteria DNA extraction kit and the fecal DNA extraction kit respectively. Then Yersinia enterocolitica and11non-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 as parallel detection at the same time.3. The fecal specimens in patients with diarrhea were collected at a community health service station and a hospital, DNA was extracted from the fecal specimens after pre-treatment and cold enrichment for18h. Optimize loop-mediated isothermal amplification(LAMP) reaction condition based on factorial experiment design. Then PCR assay and the developed LAMP method were applied in clinical faecal samples and compared to evaluate its application.Results1. The optimal combination of FIP/BIP, MgCl2, dNTPs was containing1.6uM FIP and BIP,1.6mM dNTPs,2.0mM MgCl2in25ul volumes after128LAMP reactions were completed according to4×4×4three-factor factorial design, which was confirmed by factorial experiment analysis and multiple comparisons. So the optimal LAMP condition was containing1.6uM each inner primer(FIP and BIP),0.2uM each outer primer(F3and B3),1.6mM dNTPs,2.0mM MgCl2,8U of Bst DNA polymerase,2.5ul of10×Thermopol buffer and5ul DNA template in25ul volumes. And the amplification program was as follows:63℃for60min and then heated at80℃for10min. The optimized LAMP reaction system was proved to be repeatable, reliable and stable. Results from analysis of variance displayed that there are interaction among the three factors and the order of amplification effect of the three main factors were FIP/BIP, Mg2+, dNTPs, in turn under conditions of the present study.2. 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. When the reference strain was used as the test organism, the LAMP method can detect Yersinia enterocolitica genomic DNA as low as38.05fg (observed by visual detection under UV light and normal light)/ul reaction system minimally; When detecting template DNA from pure culture bacteria, the LAMP method was capable of detecting15CFU/ml (observed by visual detection under UV light and normal light) at the least; in artificially infected faecal specimens, the detection limit of the LAMP method was150CFU/g (observed by visual detection under UV light and normal ligh).3.539fecal samples were collected from a community health service station and a hospital from diarrhea patients,13were tested to be positive using the LAMP assay by visual detection, while11of them were positive by PCR. But only11cases of positive samples of PCR were isolated and identified successfully by culture method with cold enrichment broth while2were failed from the sample of LAMP. The specificity of LAMP was calculated to be99.62%and the sensitivity of LAMP was calculated to be100.%with culture as the "gold standard". Two methods showed no significant difference (P=0.500) and had great agreementwith each other (Kappa=0.915, P=0.000).Conclusions1. Factorial design combined with variance analysis was easy to operate and time-saving in the optimization of LAMP reaction system compared with single-factor method and was more accurate, comprehensive and reliable than the simple analytical methods of direct comparison. So the novel method was a considerable method to optimize LAMP reaction system.2. The optimized constituent of LAMP including FIP/BIP, MgCl2, dNTPs proved to be sensitive, specific, and can be completed in one hour using thermostatic water bath without elaborate, expensive instruments and special reagents. Also, the amplification results can be observed by visual endpoint detection methods.3. The established LAMP method was suitable for Yersinia enterocolitica detection in faecal samples. The detection process in feces included the cold enrichment overnight, DNA extraction together with LAMP amplification and result identification, which took less than1day to be accomplished. The new method showed a satisfactory concordance with the PCR when being applied in clinical specimens.4. FIP/BIP, dNTPs, Mg2+concentrations all played important roles in LAMP amplification. There are interaction among the three factors, so optimize the LAMP with factorial experiment design is a good choose. And the order of the effects of those factors was FIP/BIP, Mg2+, dNTPs. |
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