The Establishment Of The Test Kit For The Rapid Test Of Nitrite Bacteria And Fluorescent Quantitative PCR Test

Nitrite bacteria are a kind of soil or marine bacteria that can oxidize ammonia to nitrous acid under aerobatic condition. It is the bacteria functioning at the first step of nitrification process. Nitrite bacteria widely exist in all kinds of culturing waters and can improve water conditions by changing the concentrations of ammonia nitrogen and nitride, and thus affecting culture production. Compared with the physical or chemical methods to reduce their concentration in water, this micro-biotic method is gaining increasing attention and application for its green safe and pollution-free characteristics without side-effect and drug residual.It’s the first and also the most important step of nitrification to have the AMO catalytic ammonia of ammonia oxidizing bacteria oxidized to hydroxylamine. amoA genes(Ammonia single oxygenase gene) are serving to encode the active sites of AMO in the family of ammonia oxidizing bacterial genes. Due to the different amoA genes in the different types of ammonia oxidizing bacteria, there can be clear difference in DNA levels between different amoA genes in the same family. Therefore, it is necessary to label the amoA genes as a kind of effective specific molecular for family appraisal of the ammonia oxidizing bacteria in the test sample. That explains why amoA genes are used as a target type of genes to test nitrite bacteria in the experiment.In the research, the amoA genes of the Cobetia marina(Cobetia marina DSM 4741(T), halomonas of halomonadaceae)is being taken as an example to design the Quantitative Real-time PCR specific primers, and construct the Cobetia marina fluorescence quantitative PCR detection system. The Cobetia marina fluorescence quantitative PCR detection kit is fabricated based on this system, with an aim to build a specific, sensitive and rapid nitrite bacteria detection mechanism at molecular level; thus rendering the effective combination of the rapid detection of nitrite bacteria with the research of nitrite bacteria in other fields as well as its practical production application.The experiment results are as below:First, the PCR amplification of Cobetia marina amoA gene segment. The total DNA of Cobetia marina can be successfully extracted with the help of DNA purification kit. In light of amoA gene(KGA02156) sequence of Cobetia marina bacteria, the total length of the specific amoA gene primer CmAMOF/CmAMOR is designed by means of Primer primer 5.0 software analysis.CmAMOF: 5’-CTGCTGACCTCGCTGGC-3’CmAMOR: 5’-CTATTGCGTCGCCTCACG-3’Use CmAMOF/CmAMOR to directly amplify the 1017bp-long amoA gene segment in the total Cobetia marina DNA. Use EG101 kit to recycle and purify the collected amoA gene DNA segment, and use PEASY-T3 kit to connect the purified amoA gene DNA segment to T3 carrier, and lead the connection products into Trans1-T3 competent cell for cultivation. Select the white single bacteria colony near the blue spots on the solid culture medium for positive recombinants appraisal, and adopt PCR to appraise the targeted gene introduction. Send the backteria liquid containing the target genes to a biology company for sequencing and conduct homology comparison of the sequencing results on GenBank. As a result, DNA segment and the amoA genes of the experimental bacteria share a high similarity, with the sequence identity of over 98%. Therefore, it is identified that the DNA segment obtained through amplification is the amoA genes of Cobetia marina, and thus the positive standard template CamoA-A can be obtained.Second, build Cobetia marina amo A gene fluorescent PCR system. Lead Cobetia marina bacterial amoA gene sequence into Primer 5.0 software for fluorescent PCR primer design to get the optimal primer CYamoAF/CYamoAR.CYamoAF: 5’-GCTGCTTTGCCGAACTGAT-3’CYamoAR: 5’-GTGGCGGAAATGACGTGC-3’Use CYamoAF/CYamoAR to amplify, purify, recycle the target gene segment, connect and convert the carrier before PCR appraisal and sequencing. The sequencing results are conducted homologous comparison on GenBank, and it is confirmed that the DNA segment and the amoA genes of the experimental bacteria share a high similarity, with the sequence identity of over 98%, thus confirming that the DNA fragment obtained through amplification meets the experiment requirements. Use SYBR® Premix Ex TaqTM(TaKaRa,Japan) kit to create 100-106copies/μL gradient fluorescence quantitative PCR reaction for the standard positive template, thus the standard curve equation can be obtained:y=-3.325x+39.433,R2 =0.994,linear relation is perfect; counting the initial bacterial concentration,extract bacteria gene DNA for 10 times of gradient dilution based on 1×100-1×106 for fluorescent quantitative PCR reaction. Thus the following standard curve equation:y=-3.313x+29.742,R2=0.992,linear relation is perfect. It can be concluded that the designed fluorescent PCR primer can specifically recognize the target bacteria strain with the sensitivity up to100copies/μL.Third, the Cobetia marina amoA gene fluorescent quantitative PCR kit is constructed, and the reaction conditions such as the dosage of specific primer and the reaction annealing temperature are optimized. It is figured out that the optimal dosage of the specific primer in the 20μL PCR reaction system is 0.8μL, and the best reaction annealing temperature is 58℃.Fourth, idex text of nitride bacteria amoA gene fluorescent quantitative PCR kit.Specific test: Use the kit to respectively test the pure cultured Cobetia marina bacterial solution, nitrite bacteria A(Marunomonas arenicola),nitrite bacteria B(Halomoeas ventosae), bacillus subtilis A, bacillus subtilis B, nitrobacteria A, nitrobacteria B, mixed bacteria liquid containing seven bacteria, sea water that is mixed with Cobetia marina bacterial solution, and super-pure water blank control. Among them, the target pure cultured Cobetia marina bacterial solution, the mixing bacteria liquid and the sea water that is mixed with Cobetia marina bacterial solution turn positive and the others negative. That can draw such a conclusion as the kit can specifically detect the target bacteria and the mixing bacterial containing target bacteria.Sensitivity test: the common PCR and fluorescence quantitative PCR are utilized to conduct gradient on the positive template and the bacterial solution, respectively; the test results of which indicate that the test sensitivity of fluorescence quantitative PCR is far greater than that of common PCR.Repeatability test: use the standard positive template and bacteria gene DNA template for 6-hole repeatability test, with each for five times. After data process, the fluorescent quantitative PCR variable coefficient is seen less than 2%, which suggests a good repeatability of the fluorescent quantitative kit.Stability test: after melting the reagent stored in-20°C refrigerator respectively for 1, 10, 20, 30,40 and 50 times, the fluorescent quantitative PCR variable coefficient is F=0.307 >0.05. It is found that the kit has a high stability after data process.Storage life confirmation: take out the reagent stored in-20°C temperature for fluorescent quantitative PCR test every month. The positive test results for 7 successive months suggest the Cobetia marina amoA gene fluorescent quantitative PCR test kit in the experiment can keep stable for at least 7 months.