| BackgroundClostridium difficile(C.difficile), which itself is not invasive, is Gram-positive anaerobic bacillus. Once human intestinal microenvironment is destroyed, toxin A, toxin B and binary toxins would be secreted by part of toxigenic C.difficile. It could lead to antibiotic-associated diarrhea (AAD), colitis and even fatal pseudomembranous colitis (PMC), which are referred to as C.difficile infection (CDI). With the emergence and spread of a new hypervirulent C.difficile strain (BI/NAP1/ 027), there has been reportedly an increase in the incidence and severity of CDI around the world attracting worldwide attention.Toxin A and toxin BC.difficile can be classified into toxigenic and non-toxigenic strains. Those non-toxigenic strains of C.difficile do not cause any clinical symptoms. Those toxigenic strains of C.difficile would secret toxin A and toxin B, encoded by the corresponding toxin gene tcdA and tcdB, respectively. Toxin A, referred to as enterotoxin, would tend to make ileum intestinal infiltration of neutrophils and release of lymphokines causing large number of fluid secretion and hemorrhagic necrosis. Toxin B, known as cytotoxic, would result in actin depolymerization and cytoskeletal damage. It would lead to cell shrinkage necrosis and intestinal cells damage directly. The virulence of toxin A is approximately 10 times higher than toxin B. Nowadays, it has been found toxin A-negative, toxin B-positive strains of C.difficile, while toxin A-positive, toxin B- negative strains of C.difficile have not yet been found. It indicates that toxin B alone may become pathogenic. As the global outbreak of CDI and drug resistance increasing, the recurrence rate and mortality have become significantly higher. Therefore, it is particularly important to detect C.difficile rapidly and sensitively. Choosing tcdA and tcdB as the target gene to the diagnosis of C.difficile can make comprehensive and specific identification of toxigenic strains.As the gold standard of the detection of C.difficile in feces, C. difficile cytotoxin neutralization assay (CCNA) and toxigenic culture (TC) are far away from becoming a routine clinical test because of its critical culture conditions and sophisticated technical requirements. Enzyme immunoassay (EIA) commonly used in the worldwide is rapid, specific, but not sensitive. Glutaimte dehydrogenase (GDH) is tend to be rapid and sensitive, but there are cross-reactivity, the highly false positive rate, and poor specificity. Although C.difficile can be detected by conventional PCR, it is not suitable for the rapid detection of primary hospital and field applications. Because PCR requires sophisticated equipment and complex operation. Besides, it need to electrophorese and develop the product after amplification. Therefore, another rapid, specific and sensitive method for detection of C.difficile has become a new research field in CDI.C.difficile binary toxinThere is hypervirulent C.difficile strain (BI/NAP1/027) causing widespread outbreak of CDI in North America and Europe, which secretes toxin A, toxin B, as well as binary toxin (CDT). CDT is composed of two components-cdtA and cdtB, which are located on the outside of the pathogenicity locus. cdtA is an ADP-ribose transferase, which can stop the synthesis of actin protein fragments to induce cell apoptosis. cdtB is a transport protein. After cdtB is activated by serine protease, it would bind to host cells. Then cdtB would transfer cdtA into cytolymph to induce depolymeric cytoskeleton and generate microtubules protrusions, which would enhance C.difficile colonization spontaneously.18 base pair deletion of tcdC gene are well conserved in PCR ribotype 027 isolates. The virulence of toxin A and toxin B produced by C.difficile strain (BI/NAP1/027) is 16 times and 23 times higher than traditional toxigenic strains respectively, leading to a significant increase in the mortality rate and resistance rate of CDI. Considering its outbreak frequency, risk and economic burden, it is urgent to improve the diagnosis of CDT and control its prevalence effectively.The methods to detect CDT include pulsed field gel electrophoresis (PFGE), restriction endonuclease analysis (REA), and nucleic acid testing (conventional PCR and multiplex real-time PCR). There is not any "gold standard" to detect CDT at all around the world. There are some defects of PFGE. For example, it need much time to type. Besides, it requires complicated electrophoresis conditions, special equipment and higher prices reagents. The weakness for REA is excessive DNA profiles bands and it is difficult to distinguish the result. What’s more, REA is liable to be contaminated. Although conventional PCR and multiplex real-time PCR is the most commonly used detection of CDT in our country, it is not suitable for the rapid detection of primary hospital and field applications. Because it requires sophisticated equipment and complex operation. Besides, it need to electrophorese and develop the product after amplification. Therefore, in order to early screening of hypervirulent strains, it is necessary to develop another rapid, specific and sensitive method for detection of CDT contributing to controlling its prevalence effectively and guiding the clinical treatment for CDI.Loop-mediated isothermal amplificationLoop-mediated isothermal amplification (LAMP) assays aim at six or eight distinct sequences on the target DNA to design four or six specific primers under isothermal conditions (63-67℃). LAMP assays rely on auto-cycling strand displacement DNA synthesis in the presence of Bst DNA polymerase to produce a large number of target DNA and a white precipitate of magnesium pyrophosphate simultaneously, resulting in the rapid detection of the target gene. The assays require generally shorter time (30-60 minutes) and are less labor-intensive. Besides, the equipment of the assays is not sophisticated that a temperature-controlled water bath or another heat-stable device is sufficient. With the help of chromogenic reagent, LAMP has become an approach to directly visualize the reaction. Nowadays, LAMP has been widely used in the clinical diagnosis of epidemic bacteria, viruses, parasites, and fetal sex identification.Purpose and significanceThis study aims to develop a rapid, convenient, specific, sensitive and visual method for detection of tcdA, tcdB, cdtA and cdtB. The developed assays are suitable for the primary health care units and Centers for Disease Control and Prevention to screen C.difficile and its hypervirulent strains. It also provides a new technology platform for the detection of C.difffiile and its toxin genotyping, contributing to controlling its prevalence effectively and guiding the clinical treatment for CDI.Research methods and contentSubjects:A total of 156 stool samples were collect from patients with diarrhea in Nanfang Hospital from August 1,2013 to January 31,2014 (5-7 levels in Bristol classification) and analyzed for tcdA, tcdB, cdtA and cdtB of C. difficile. Excluding the discarded specimens for technical factors,123 samples were suitable for the study of CDI.Methods:1). Rapid detection of tcdA and tcdB gene in C.difficile by LAMP.① LAMP primers were designed to target six or eight distinct sequences downloaded from the NCBI GenBank database. Primer Explorer V4 software was chosen to design the primers, targeting tcdA and tcdB gene, respectively.②To determine the most appropriate primer, the set was chosen by amplification efficiency.③To determine the most appropriate temperature of the LAMP reaction, different temperatures were tested in the range from 58℃ to 69℃ at 1℃ intervals using the optimal primer set. Using the DNA of C.difficile VPI10463 as a template, the appropriate temperature was selected by the fastest amplification of the target sequence.④To determine the LAMP assay limit, genomic DNA was extracted from C.difficile VPI10463 as a template, and then serially diluted 10-fold from 207 ng/μl to 0.000207 pg/μl Distilled water was used as negative control. The sensitivity of the primers was detected by both LAMP and PCR. Real-time monitoring of turbidity and chromogenic reaction were used to determine negative and positive results. ⑤The C.difficile VPI10463 strain as positive control was used to estimate the specificity of the LAMP reaction for detecting tcdA and tcdB. Twenty-six enteric pathogenic bacterial strains of different species without tcdA and tcdB as well as distilled water were designed as negative controls. Real-time monitoring of turbidity and chromogenic reaction were selected to determine results. ⑥Comparision of the efficiency between LAMP, PCR and PCR-based sequencing for detection of tcdA. Comparision of the efficiency between LAMP, PCR and CCNA for detection of tcdB.2). Rapid detection of cdtA and cdtB gene in C.difficile by LAMP. ①LAMP primers were designed to target six or eight distinct sequences by Primer Explorer V4 software, targeting cdtA and cdtB gene, respectively. ②To determine the most appropriate primer, it was selected by the fastest amplification of the target sequence. ③To determine the most appropriate temperature of the LAMP reaction, different temperatures were tested in the range from 57℃ to 64℃ at 1℃ intervals for cdtA detection. Different temperatures were set in the range from 57℃ to 68℃ at 1℃ intervals for cdtB detection. Using the DNA of C.difficile ribotype 027 as a template, the appropriate temperature was chosen by amplification efficiency. ④ To determine the LAMP assay limit, genomic DNA was extracted from C.difficile ribotype 027 and then serially diluted 10-fold from 24.8 ng/μl to 0.000248 pg/μl Distilled water was used as negative control. Both LAMP and PCR was used to determine the sensitivity of the primers at the same time. Real-time monitoring of turbidity and chromogenic reaction were including to determine results. ⑤ The C.difficile ribotype 027 strain was used as positive control to estimate the specificity of the LAMP reaction.Twenty-five enteric pathogenic bacterial strains of different species as well as distilled water were designed as negative controls. Real-time monitoring of turbidity and chromogenic reaction were selected to determine results. ⑥The LAMP assay was compared to conventional PCR with 123 clinical isolates for detection of cdtA and cdtB. Research processResults:1). The A12 primer set amplified the target sequence faster than the other primer sets; therefore, it was selected as the most appropriate primer set for tcdA detection by LAMP. The optimal reaction conditions for tcdA detection were at an isothermal temperature of 61℃ within 60 min. The most appropriate reaction conditions for tcdB detection were at an isothermal temperature of 60℃ within 60 min using the BO primer set. The results of the 26 bacteria strains were negative, which exhibited the high-specificity of the primers. The detection limit of LAMP detecting tcdA and tcdB was 20.7 pg/μl DNA, which was 10-fold greater than that of PCR. The positive rate of tcdA in C. difficile from 123 stool specimens was 29.3 by PCR-based sequencing. While the specificity between LAMP and PCR was no significant difference, the sensitivity and the consistent percentage of the LAMP method were better than PCR. The positive rate of toxin B in C. difficile from 123 stool specimens was 25.2% by CCNA. There were no statistics differences between the LAMP method and the PCR method when detecting the sensitivity and specificity of toxin B in C. difficile.2). The most appropriate reaction conditions for cdtA detection were at an isothermal temperature of 60℃ within 90 min using the A2 primer set. As the B4 primer set amplified the target sequence faster than the other primer sets, it was selected as the most appropriate primer set for cdtB detection by LAMP. The optimal reaction conditions for cdtB detection were at an isothermal temperature of 63℃ within 90 min. The most appropriate reaction conditions to detect cdtA and cdtB at the same time were within 90 min at an isothermal temperature of 61℃. The results of the 25 bacteria strains were negative, indicating the high-specificity of the primers. The detection limit of LAMP detecting cdtA and cdtB was 24.8 pg/μl DNA, which was 10-fold greater than that of PCR. The cdtA gene and cdtB gene were detected in 2.44% (3/123) of the clinical isolates by both LAMP and conventional PCR.ConclusionThe developed LAMP method is exhibited to be a potentially valuable, rapid, specific, sensitive, and visual assay for the rapid detection of tcdA, tcdB, cdtA and cdtB respectively. |
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