Research And Application On Point-of-Care Testing Of Clostridium Difficile

Clostridium difficile is a strictly anaerobic gram-positive bacterium,which is widely present in the natural environment and human and animal intestines.It is generally believed that this bacterium is the main cause of antibiotic-related diarrhea and pseudomembranous colitis.Pathogenic bacteria are the primary cause of antibiotic-associated diarrhea（AAD）in hospitals.In recent years,due to the popularity and irregular use of antibiotics,and the emergence of highly pathogenic strains BI/NAP1/027,the incidence of Clostridium difficile infections（CDIs）has continued to increase;at the same time,the resistance of Clostridium difficile has also increased.Increasingly serious,the infection of Clostridium difficile poses a serious threat to human life and health worldwide,and the need for rapid diagnosis of CDIs is particularly urgent.At present,the diagnostic methods of CDIs mainly include cytotoxicity detection,toxin-producing bacteria culture,enzyme-linked immunosorbent assay,glutamate dehydrogenase detection,and nucleic acid amplification detection,but there is no recognized single detection method as a CDI diagnosis method.Many experts support the use of two-step or three-step diagnosis of CDI to improve specificity and sensitivity,which will inevitably lead to delays in disease diagnosis and affect patient infection control.In view of this,this paper uses the main virulence factor of Clostridium difficile toxin B（TcdB）as the detection target,and conducts rapid detection technology research.In this thesis,the non-toxic TcdB protein mutant a TcdB constructed in the early stage of our research group was used as the antigen to immunize mice to prepare hybridoma cells that can secrete anti-TcdB antibodies.Finally,four positive hybridoma cell lines with higher titer were screened（4B9,3G7,3C6,1E6）,the corresponding monoclonal antibodies were obtained by preparing ascites.The titers and affinities of the four antibodies were analyzed by indirect ELISA and molecular interaction methods.4B9,3G7,3C6,and 1E6 were all Ig G1 type;the antibody titers were 1×10⁵、1×10⁶、1×10⁶and 1×10⁶,respectively.The affinity constants are2.17×10^-8、1.38×10^-8、3.03×10^-9and 1.03×10^-8,respectively.The establishment of TcdB automatic chemiluminescence immune assay（CLIA）:According to antibody pairing screening,the antibody pairs 3C6 and 1E6 with magnetic beads and alkaline phosphatase,respectively,to establish a CLIA detection system.The detection range is 0.12～150 ng/m L,and the limit of detection（LOD）is 1.83 pmol/L.The recovery rate is between 97.3%and 103.25%,and the coefficient of variation between different batches is less than 15%,which meets the clinical testing standards.Compared with the commercial ELISA kit,the correlation coefficient between the two methods is 0.9893,and the average relative difference is only 3.8%by Bland-Altman analysis.The detection time has been shortened from 3 hours for commercial ELISA kits to 30 minutes.The establishment of TcdB fluorescence quantitative immunochromatographic detection method:after optimization,the anti-TcdB antibody concentration is 400μg/m L,and the p H 6.0MES solution is used as the buffer,and the EDC and NHS are activated for 20 minutes and then coupled for 2 hours to prepare the fluorescence.Microsphere probe.A fluorescence quantitative immunolayer detection system was developed,and the application of this method was evaluated in combination with a commercial kit.The results showed that the correlation coefficient of the standard curve of TcdB fluorescence immunochromatography was 0.9896,and the chromatography time was 15 minutes.The LOD is as low as 1.79 pmol/L,the detection range is 0.4-150 ng/m L,the average recovery is between 92.6%and 107.3%,and the coefficient of variation between different batches is less than 10%,which meets the clinical testing standards.Compared with the commercial ELISA kit,the correlation coefficient of the detection results of the two methods was 0.9881,and the average relative difference was only 4.16%by Bland-Altman analysis.TcdB quantum dot immunochromatographic detection method was established:after optimization,MES solution with anti-TcdB antibody concentration 150μg/m L and p H 6.0 was used as buffer solution,and EDC and NHS were used as activators for 30 min and then coupled for 2 h.Quantum dot immunoprobe.A quantum dot quantitative immunolayer detection system was developed,and the application of this method was evaluated in conjunction with a commercial kit.The results showed that the correlation coefficient of the standard curve of TcdB quantum dot immunochromatography was 0.9902,and the chromatography time was 12min.The LOD is as low as 1.1 pmol/L,the detection range is 0.2-150 ng/m L,the average recovery is between 95.58%and 112.5%,and the coefficient of variation between different batches is less than 10%,which meets the clinical testing standards.Compared with the commercial ELISA kit,the correlation coefficient of the detection results of the two methods was 0.9915,and the average relative difference was only 3.33%by Bland-Altman analysis.The establishment of a quantum dot nucleic acid detection method for Clostridium difficile:5 specific genes were screened through the comparison of 1484 Clostridium difficile genomes,and the CD630DERM＿RS02920 gene and the virulence gene TcdB were selected to carry out the combined detection of the toxin-producing bacteria of Clostridium difficile.After optimization,the anti-FAM antibody was coupled to the self-made quantum dot microspheres,and the MES solution with a concentration of 600μg/m L and p H 6.0 was used as the buffer solution.After 15 minutes of EDC and NHS activation,the coupling was coupled for 2.5 hours.After amplifying for 20 min at 39 ^oC,diluted in a ratio of 1:200,the test result can be obtained after chromatography on the self-made reagent strip for 5 min,and the LOD is as low as 100fg/μL.Evaluating the detection performance of the above detection system/platform,it is found that the CLIA detection system has the advantages of rapidness,high sensitivity,and high throughput,which can be applied to hospital clinical diagnosis and treatment scenarios.In addition,the two detection platforms of fluorescence quantitative chromatography and quantum dot immunochromatography have shown good consistency with the existing commercial toxin B detection kits.Because of their fast,simple,high sensitivity,low price and other advantages,they can satisfy general community hospitals and home testing needs.The method based on quantum dot nucleic acid detection of C.difficile as a new detection platform shows a relatively excellent lower limit of detection,and the cost is much lower than that of traditional nucleic acid detection methods and does not rely on specific instruments.Combining with the aforementioned three detection methods can effectively improve the detection of C.difficile efficiency,in addition to community hospitals and home scenarios,it is also applicable to on-site testing requirements in resource-poor areas.