Establishment And Application Of LAMP Method For Detection Of AmpC Gene In E.coli

1. Background and purposeIn recent decades, bacterial resistance has developed into a serious public health crisis. It resulted in the patients with treatments failure, medical expenses and mortality rising. Even more serious, the further development of drug-resistant could make human re-face the threat of infectious diseases. World Health Organization （WHO） recently warned that if human beings do not act quickly and effectively, bacterial resistance crisis will fully come. At present, bacteria can become resistant to almost all antibiotics, among all, the resistance caused by β-lactamases is one of the most worrying examples. Nowadays, the β-lactamases have evolved from ordinary enzymes to the broad-spectrum enzymes, ESBLs, carbapenemases etc. Escherichia coli and Pseudomonas aeruginosa resistant to all β-lactam antimicrobial drugs have been found in clinical.AmpC β-lactamase is one kind of β-lactamases generated mainly by Enterobacteriaceae, Citrobacter, Pseudomonas aeruginosa etc. It can hydrolyze penicillin, the first, second, third generation cephalosporins and monocyclic β-lactam antibiotics, also cause resistant to many kind of β-lactam antibiotics at one time or on one patient. In recent years, with the widespread use of cephalosporins and β-lactam antibiotics, the AmpC β-lactamases climcal detection rate is rising all the time, and it has become the main cause of resistance in Gram-negative bacilli.The synthesis of AmpC P-lactamase relates to5genes, they are ampC、ampR、 ampD、ampE and ampG ampC gene is the structural gene of AmpC β-lactamase, it mostly presents in the bacterial chromosome, sometimes in the plasmid. It can encode the AmpC β-lactamase whose molecular weight is39.6KDa, composed of380amino acids. Plasmid-mediated AmpC β-lactamase is different from chromosome-mediated AmpC β-lactamase, it can encode AmpC β-lactamase continuously and high-yield without antibiotics induced. Plasmid-mediated AmpC P-lactamase can also spread rapidly to a different genus and species, resulting in serious bacterial resistance.Now, the methods for detection of AmpC β-lactamase mainly could be summarized into2ways:The first way is extracting the AmpC P-lactamase in bacteria, to make the drug sensitive test, such as cefoxitin susceptibility testing, AmpC phenotype screening test, flucloxacillin （FCC） double disk diffusion collaborative experiment, three-dimensional test and so on. The other way is to detect the coding genes of AmpC β-lactamase. The methods detecting AmpC P-lactamase directly have high reliability, but they need to extract the AmpC β-lactamase from bacteria, often are tedious and time-consuming, so they can not become the routine ways used in most clinical microbiology laboratory. The ways to detect genes of AmpC P-lactamase are using molecular biology techniques to detect wether the genes of AmpC β-lactamas are exist, they could have accurate results, but need to strictly prevent false positive and false negative results due to pollution.Loop-mediated isothermal amplification （LAMP） is a new nucleic acid detection technology established by Japanese scholars Notomi and his colleagues. It designs4or6pairs of special primer according to the target genes6or8region, then uses a chain replacement DNA polymerases （Bst DNA polymerase） in isothermal conditions （about65℃）, and keeps the temperature for dozens of minutes, then the nucleic acid amplification reaction can complete. LAMP is a new nucleic acid amplification method, which is simple, fast, strong specific, compared with conventional PCR, it does not require a template thermal denaturation and temperature cycling processes, any specialized equipment is not need either, can be used to detect anywhere. The sensitivity, specificity, and detection range of LAMP are comparable to or even better than the PCR technique, its detection cost is far lower than quantitative PCR, suitable for large-scale sample testing, and now is widely used in disease diagnosis, pathogenic microorganism inspection and the animal embryos in the gender identification, etc.The ingredients of LAMP reaction system mainly includes DNA template, the primers （including the inner primers, the outer primer and loop primer）, betaine, dNTPs, and Mg2+, Bst DNA polymerase, Bst buffer etc. In order to detect the target gene accurately with LAMP assay, optimizing the reaction system and making out the optimum concentration of the various ingredients are necessary. After the reaction system established, the sensitivity and specificity of the whole method need to be evaluated. Because LAMP has high sensitivity, it needs to take appropriate measures to prevent pollution during the trial, in order to avoid causing false-positive results.Escherichia coli are always recognized as the pathogen with the highest infection rate, not only that, infectious bacterial resistance monitoring results of Chinese hospital show that, Escherichia coli resistance rates increased year by year. Especially over the last decade, with the widespread use of broad-spectrum β-lactam antibiotics, especially the third-generation cephalosporin, the Escherichia coli producing AmpC β-lactamase is increasingly common. Escherichia coli produce AmpC β-lactamase could make them resistant to many kinds of antibiotics, which include penicillin and nearly all cephalosporin except cefpirome and cefepime. In addition, it has been reported that some strains of Escherichia coli can produce both AmpC β-lactamase and ESBLs, broad-spectrum cephalosporin-resistant Escherichia coli is reported too. They not only lead to treatment failure, but also carry other resistance gene, also limits the treatment options.Establishing the system of screening the bacteria producing AmpC β-lactamase and making clear the prevalence of them are the preconditions of developing effective countermeasures. This study aims to establish the LAMP assay for detection of ampC gene of Escherichia coli, verify its validity when applied to detect the E.coli which are producing AmpC β-lactamase, and initially explore the application value of detecting ampC gene.2. MethodsEstablishment of LAMP method for detection of AmpC β-lactamase of E.coli（1） According to the conservative district of ampC gene sequences （No: NC₀08490.1） of E.coli, we designed a set of LAMP primers, which include two outer primers F3and B3, two inner primers F3and B3, two Loop primers LB and LF with primer design software Primer Explorer4.0, established a25ul LAMP reaction system.（2） Optimized the LAMP reaction system through agarose gel electrophoresis, determined the concentrations of dNTPs, betaine, Mg2+, as well as the best reaction time.（3） Verify the specificity and sensitivity of LAMP established by us, and compared it with PCR, to find the advantages of the LAMP method.The clinical value of detecting AmpC β-lactamase of E.coli with LAMP（1） Extracted the genomic DNA of54strains of E. coli come from clinic by boiling method, detected the ampC gene with LAMP and PCR.Extracted the plasmid of54strains of E.coli, detected the ampC gene using LAMP, calculated the proportion of plasmid-mediated AmpC β-lactamase.（2） According to the susceptibility testing standards of CLSI, detected the AmpC P-lactamase of54strains of E.coli. The criterion is when the inhibition zone diameter is less than18mm, see it as AmpC β-lactamase positive.（3） Thawed and refroze the54strains of E.coli to extract the AmpC β-lactamase, Three-dimensional test screened the AmpC β-lactamase positive strains.（4） Compared the three methods above-mentioned with statistical methods, evaluated the clinical value of LAMP on the detection of drug resistance genes.3. Results（1） Finally we make sure the LAMP reaction system contains:2μl DNA template,1.6μM inner primers（FIP、BIP）,0.4μM outer primers（F3、B3） and loop primers（LF、LB）,2.5μl10×Bst buffer,0.6mM dNTPs,1.0M betaine,12mM MgCl2,8U Bst DNA polymerase, add ddH2O to25μl. When keep the system in65℃for60min, the amplified product could be observed as white precipitation by the naked eyes,if analyzed on2%agarose gel electrophoresis, typical LAMP ladder bands could be observed.（2） The specificity of LAMP is identical with PCR, and its sensitivity is about10times higher.（3） LAMP and PCR both detected41strains of E.coli which carry ampC gene,13strains didn’t. There are21strains of E.coli whose plasmids carrying ampC gene, accounted for38.89%of all E. coli.（4） The cefoxitin susceptibility testing screened32strains of E.coli sensitive to cefoxitin,22strains are drug-resistant. The three-dimensional test discovered19 strains of E.coli produced AmpC β-lactamase,35stains did not.（5） The statistical analysis showed that there was significant difference between LAMP and cefoxitin disk susceptibility test（P=0.000）, and their goodness of fit was weak（K=0.358）; there was significant difference between LAMP and three-dimensional test（P=0.000）, their goodness of fit was weak too（κ=0.294）; but there was no statistical difference between the cefoxitin disk susceptibility test and three-dimensional test（P=0.25）,their goodness of fit was strong（κ=0.882）.4. Conclusion（1） Established the LAMP method to rapidly and accurately detect the ampC gene of E.coli, it is suitable for application at grass roots.（2） The13strains of E.coli which were ampC gene-negative detected by LAMP, were all AmpC P-lactamase-negative detected by cefoxitin susceptibility test and three-dimensional test; The22strains which were resistant to cefoxitin and the19strains which were AmpC P-lactamase-positive detected by three-dimensional test, were all ampC gene-positive;The strains which were ampC gene-positive but AmpC β-lactamase-negative,if contact with antibiotics, maybe begin to express AmpC β-lactamase. These showed that using LAMP to detect the ampC gene, can guide the clinic choosing appropriate antibiotic drugs, so that decrease the bacterial drug resistance due to misuse and abuse antibiotic drugs.（3） LAMP can used to detect plasmids whether carrying the ampC gene, which can contribute to make out the speed of the ampC gene’s spread and monitor its dissemination trends.