Product introduction： The MA qPCR live bacteria detection kit provides an effective means for detecting bacterial activity. The kit provides a mixture of PMA dye and qPCR based on SYBR Green dye. The optimal amount of dye and the number of samples that can be treated may vary depending on the type of sample. PMA is a high-affinity DNA-binding dye, especially with double-stranded DNA. The dye itself has weak fluorescence, but it can emit brighter fluorescence after binding to nucleic acids. PMA is impermeable to cell membranes, so it can selectively modify the DNA of dead cells with damaged membranes. After the PMA-modified DNA is photolyzed by blue light ( ~ 464 nm ), the photoreactive azide group on the PMA is converted into a highly reactive nitrene radical, which reacts with any hydrocarbon near the DNA binding site to form a stable covalent nitrogen-carbon bond, resulting in permanent DNA modification. This modification process will make DNA insoluble and lost with cell debris during the later genomic DNA extraction process. The unbound PMA remaining in the solution reacts with water molecules under strong light irradiation to decompose into hydroxylamine compounds without cross-linking activity, so that it can no longer covalently bind to DNA. Based on this feature of PMA, PMA was combined with qPCR technology to form a new detection method, PMA-qPCR, for the screening of live bacteria. At present, the method has been verified in a variety of bacterial strains, yeast, fungi, viruses and parasites. The treatment of complex samples, such as manure or soil, may require optimization of sample dilution, dye concentration, and light treatment time. The treatment of diluted samples, such as water testing, may require filtration or concentration prior to dye treatment.
Matters needing attention： 1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. the components of the kit contain fluorescent dyes. Avoid light during use and storage. 3. for your safety and health, please wear experimental clothes and disposable gloves. Product parameters:
Spectral characteristics :PMA: Ex = 464 nm; Ex/Em = 510/610 nm (following photolysis and reaction with DNA/RNA) Component：
PMA： Ex = 464 nm; Ex/Em = 510/610 nm (following photolysis and reaction with DNA/RNA)
Instruction：
Precautions before use： 1.This live bacteria detection kit distinguishes dead bacteria and live bacteria according to cell membrane permeability. Many methods of killing bacteria cause damage to the cell membrane and are therefore compatible with this kit. But some methods, such as ultraviolet irradiation, may not immediately cause cell membrane rupture. Therefore, before selecting this kit, it is necessary to carry out literature search and pre-experiment to determine whether the kit is suitable for the bacterial type and killing method you choose.
2.After PMA treatment, the bacteria need to be photolyzed to covalently bind the dye to dead cell DNA. Photolysis operations can use blue or white light sources. Generally speaking, the brighter the lamp, the higher the efficiency of the photolysis step. Non-LED lamps ( such as halogen lamps ) may heat your sample and have a negative impact on the analysis. Ice is required to cool the sample during irradiation. 3.Sample can be cryopreservation after photolysis. Frozen samples before PMA treatment photolysis may damage the cell membrane and produce false negative results. If the sample needs to be frozen before detection, it is recommended to perform a pre-experiment first.
4.Part of the mechanism of PMA is to remove PMA covalently modified DNA from the sample by precipitation ; therefore, when extracting genomic DNA, it is necessary to use the same volume of genomic DNA eluent for volume normalization. The positive control can use the genomic DNA of living cells. 5.In order to verify the effectiveness of PMA in the test sample, the Ct ( dCt ) changes between- / + PMA can be compared.
Experimental materials ( self-provided )： ①Light source ( for the photolysis step after PMA modification of DNA ) ; ② Bacterial genomic DNA extraction kit ; ③ effective qPCR primers corresponding to the sample type
Experimental procedure：
1.Suck 10 μL of E.coli bacterial solution in liquid LB medium, and culture E.coli in the bacterial incubator overnight or longer to the logarithmic growth phase ( OD600 ≈ 1.0 ) ; Note : The culture time is adjusted according to the experiment. 2.Two portions of live E.coli, 400 μL each, were placed in a clean centrifuge tube ; 3. ( Recommended ) Preparation of dead E.coli. If the dead E.coli is needed as a control, the dead E.coli can be obtained by heating the living E.coli in a water bath at 95 °C for 5 min, or at 58 °C for 3 h. the subsequent operation of the dead E. coli is the same as that of the living E. coli ; 4.Two copies of live E.coli, one without PMA treatment, and one with 25 μM PMA treatment ( the optimal PMA concentration for treating different types or different sources of bacteria needs to be consulted in the relevant literature ) ;
5.The PMA-treated samples were placed on a shaker at room temperature and incubated in the dark for 10 min to fully mix the dye with the sample ;
6.Exposure of the sample, you can use blue or white light source, irradiation time to explore their own. For example, a 60 W blue light can be used for 15 min.
Note : 1 If a halogen lamp is used, we recommend that the PMA-treated sample tube be placed on an ice block 20 cm away from the light source. Ice should be placed in a transparent tray. Adjust the light source to point directly to the sample, photolysis for 5-15 min ; if the bacteria obtained from the environment are directly used for experiments, due to the complexity or turbidity of the environmental samples, the photolysis time needs to be prolonged appropriately.
7.Treated and untreated live E.coli 5000 × g, centrifuged for 10 min, remove the supernatant ;
8.Select the appropriate genomic DNA extraction kit according to the sample type, and use the same elution volume for each group of samples when elution DNA.
Note : DNA extraction steps refer to the instructions of the kit used. Part of the mechanism of action of PMA is to remove PMA-bound DNA from the sample by precipitation ; therefore, when extracting genomic DNA, each group should use the same volume of genomic DNA eluent for volume normalization ( the amount of genomic DNA extracted from dead bacteria and live bacteria is inconsistent, so the concentration of the two is significantly different ).
9.Preparation of reaction mixture according to the following system : 
Note : 1 For the DNA extracted by commercial DNA extraction kit, the qPCR template was optimized with 2 μL as the initial volume ; 2 The template volume should not exceed 10 % of the final reaction volume ; 3 Template concentration : gDNA as template, usually 1-10 ng ; the final concentration of PCR primers is usually 0.4μM, which can get better results. When the reaction performance is poor, the primer concentration can be adjusted in the range of 0.2-1μM.
10.Slightly vortex the reaction mixture, transfer the fixed volume to the PCR tube.
11. Test procedure 
Note : 1 The extension time is adjusted according to the instrument ; the Taq enzyme in mix can be activated within 2 min, but the genomic DNA may require longer denaturation time, which can be increased at this time, and the specific denaturation time can be adjusted according to the sample type. 12. ( Optional ) Data analysis
Using live bacteria and dead bacteria as controls, the number of live cells in the sample was analyzed and calculated. It is recommended to verify the suitability of primers and PCR procedures before starting PMA qPCR detection of live bacteria.
Calculation of dead and living bacteria control dCt ( 1 ) After the end of qPCR, the Ct value of each sample was calculated by instrument software ; ( 2 ) By calculating the dCt of each control bacteria, it was judged whether PMA successfully inhibited the amplification of dead bacterial DNA. The calculation is as follows :
dCt live = Ct ( live, PMA treated ) -Ct ( live, PMA untreated ) dCt die = Ct ( die, PMA treated ) -Ct ( die, PMA untreated )
( 3 ) The dCt expectation of living bacteria is close to 0 ± 1, which indicates that PMA does not affect the amplification of living cell DNA ; ( 4 ) The expected value of dCt of dead bacteria is greater than 4 ( dCt is 4 means that it is reduced by about 16 times, that is, 94 % of dead bacterial DNA is removed ; a dCt of 8 indicated a decrease of about 250 times, that is, 99.6 % of the dead bacterial DNA was removed ).( 5 ) The dCt of dead bacteria depends on many factors, including : strain / cell type ; the way bacteria are killed ; the concentration of PMA used ; amplified sequence length.
13. Calculation of the proportion of viable ( optional ) bacteria
If the control results of dead and live bacteria are normal, the proportion of live bacteria in the sample can be calculated. ( 1 ) Calculate the dCt value of the sample :
dCt sample = Ct ( sample, PMA treated ) -Ct ( sample, PMA untreated ) ( 2 ) Conversion of dCt value to live bacteria ratio :
PMA inhibition multiple = 2 ( sample dCt )
Viable bacteria % = 100 / PMA inhibition multiple
14. ( Optional ) Calculate the absolute number of live bacteria
If you want to calculate the absolute number of viable bacteria in the sample, you need to use a known number of target bacteria genomic DNA to make a standard curve. It is recommended that the diluted concentrations of several groups of genomes are within the range of the qPCR analysis system. ( 1 ) qPCR was performed with the appropriate genome, and the Ct value was used as the ordinate, and the number of cells was used as the abscissa. The R2 value is calculated to determine the linearity, and the slope and y-axis intercept are displayed. ( 2 ) Calculate the copy number of the experimental samples :
Ct = slope * cell number + y axis intercept ( y = mx + b )
Bacterial count sample = ( Ct-y axis intercept ) / slope
Note : The live bacterial DNA was not lost during the purification process.
Examples :
Scope of application： Live bacteria detection | 
K2422234