Summary of the invention
The purpose of this invention is to provide a kind of HBV nucleic acid rapid extraction reagent, it adopts the single stage method operation to finish the quick release extraction of HBV DNA in biological sample serum, the blood plasma, the nucleic acid that obtains is applicable to detected downstream such as fluorescent PCR, has easy and simple to handle, use safety, advantage with low cost is adapted at applying in the clinical gene test.
Technical scheme
The invention provides a kind of HBV nucleic acid rapid extraction reagent, it contains 1-5%KCl (mass/volume ratio, w/v), 0.01-0.5mg/ml surfactant peptides, 0.1-2%Triton X-100 or Tween 20 (quality/plasmid ratio, v/v) and 0.001-0.005% malachite green (w/v), described quality or volume percent serve as to calculate benchmark to extract reagent volume.
HBV nucleic acid rapid extraction reagent compound method of the present invention is: adding KCl final concentration reaches 1-5% (w/v) in the sterilization deionized water, adding surfactant peptides final concentration reaches 0.01-0.5mg/ml, adding TritonX-100 or Tween 20 final concentrations reach 0.1-2% (v/v), adding malachite green final concentration reaches 0.001-0.005% (w/v).The reagent of preparation can be in room temperature preservation.
HBV nucleic acid rapid extraction reagent using method of the present invention is: get the nucleic acid rapid extraction reagent of above-mentioned preparation, add in the pending biological sample of equal-volume, with 5-10 mixing of pipettor piping and druming, HBV DNA namely extracts from virion and discharges.Described biological sample is serum, blood plasma.Extract after reagent and the sample mix liquid and transferred to colourlessly by blue-greenish colour, can be used as template and directly add the PCR reaction solution and carry out augmentation detection.
Nucleic acid rapid extraction reagent of the present invention adopts protein denaturant and sanitas, and energy rapid damage HBV virion coat protein structure discharges HBV DNA, and extraction reagent contains the component that suppresses nuclease and follow-up PCR experiment do not exerted an influence; Extract add sample in the reagent after color change, whether avoid causing obscuring of application of sample.Adopt HBV nucleic acid rapid extraction reagent of the present invention and at present method commonly used relatively need not heating, centrifugally operated, only need a pipettor at ambient temperature single stage method finish the extraction of HBV DNA in the biological sample.The nucleic acid that obtains can be widely used in fields such as follow-up fluorescent PCR detection by quantitative, the detection of PCR-gene chip hybridization.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can do various technical changes or modification to the present invention by technology general knowledge after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Main agents related among the present invention illustrates:
KCl is analytical pure, and tensio-active agents such as Triton X-100 and Tween 20 are biological level purity, and malachite green is chemical pure, and mentioned reagent is available from Chemical Reagent Co., Ltd., Sinopharm Group, and surfactant peptides is available from Sigma.Highpure Total Nucleic Acid Kit (silica gel embrane method) used in the embodiment is available from Luo Shi diagnostic products (Shanghai) Co., Ltd.; Platform is moulded DNA/RNA extraction agent (paramagnetic particle method) available from Taisushengyi Science Technology Co., Ltd.; Hepatitis B virus (HBV) nucleic acid fluorescent quantitative PCR detection kit is Shanghai clone Biology high technology Ltd's product.
Embodiment 1: and the alkaline lysis boiling method relatively is used for the detection of serum HBV DNA extraction fluorescent PCR
(1) HBV nucleic acid rapid extraction reagent preparation
According to summary of the invention, at first prepare HBV nucleic acid rapid extraction reagent: adding KCl final concentration reaches 2% (w/v) in the sterilization deionized water, adding surfactivity peptide concentration reaches 0.5mg/ml, adding Triton X-100 or Tween 20 concentration reach 0.5% (v/v), adding indicator malachite green concentration reaches 0.001% (w/v).
(2) HBV positive serum nucleic acid extraction
To 5 routine HBV positive serums, adopt the extracting method in the summary of the invention to handle: get 3.5 μ l and extract reagent adding 0.2ml pcr amplification pipe, add 3.5 μ l serum respectively, blow and beat 5-10 time repeatedly with pipettor, the operation required time is 5min.
Simultaneously, boiling extraction HBV DNA method with traditional virus precipitation-alkaline lysis in hepatitis B virus (HBV) the nucleic acid fluorescent quantitative PCR detection kit is contrast: respectively get 50 μ l serum, add 50 μ l nucleic acid extraction liquid A respectively, 10 seconds kinds of vibration mixing, 13, centrifugal 10 minutes of 000rpm abandons supernatant; Add 50 μ l nucleic acid extraction liquid B respectively to precipitation, vibration 10 seconds of mixing, 100 ℃ of boiling water baths 10 minutes, centrifugal 2 minutes of 13,000rpm, the operation required time is 30min.
(3) HBV DNA fluorescence quantitative PCR detection
Test kit HBV PCR damping fluid n * 30 μ l, MgCl got in two kinds of HBV DNA HBV nucleic acid fluorescent quantitative PCR test kit detections that method is extracted
2N * 5 μ l, HBV fluorescent probe n * 5 μ l, Taq enzyme n * 3 μ l mix in a centrifuge tube (n is sample number to be amplified), and the vibration mixing is 10 seconds on the vortex vibrator, and the low-speed centrifugal several seconds is by every pipe 43 μ l packing PCR reaction solutions.Nucleic acid rapid extraction reagent of the present invention is extracted the HBV DNA that obtains, as long as the PCR reaction solution is added above-mentioned containing in the amplification pipe that extracts reagent and serum mixed solution; To contrast method, then draw in the PCR reaction solution amplification pipe that 7 μ l templates add 43 μ l packing.
Carry out augmentation detection at ABI7500 amplified fluorescence instrument, program is: behind 50 ℃ of 2min, the 94 ℃ of 5min according to 93 ℃ of 30 seconds → 60 ℃ 90 seconds cyclic amplifications 40 times.Reaction finishes the back and uses the instrument software analytical results, and two kinds of methods are to 5 routine serum HBV DNA quantitative result such as tables 1, and two kinds of method quantitative results are close as can be known, and the method operation of reagent is easier, the time is shorter, has advantage but employing the present invention extracts.
Table 1 and alkaline lysis boiling method relatively are used for serum HBV DNA extraction fluorescent PCR detected result
Embodiment 2: and post method, paramagnetic particle method relatively are used for the detection of blood plasma HBV DNA extraction fluorescent PCR
(1) HBV nucleic acid rapid extraction reagent preparation
According to summary of the invention, at first prepare HBV nucleic acid rapid extraction reagent: adding KCl final concentration reaches 5% (w/v) in the sterilization deionized water, adding surfactivity peptide concentration reaches 0.01mg/ml, adding TritonX-100 or Tween 20 concentration reach 2% (v/v), adding indicator malachite green concentration reaches 0.004% (w/v).
(2) the positive blood plasma nucleic acid extraction of HBV
To the positive blood plasma of 5 routine HBV, adopt the extracting method in the summary of the invention to handle: get 3.5 μ l and extract reagent adding 0.2ml pcr amplification pipe, add 3.5 μ l serum respectively, blow and beat 5-10 time repeatedly with pipettor, the operation required time is 5min.
Simultaneously, moulding living doctor's DNA/RNA extraction agent (paramagnetic particle method) processing HBV positive blood paste-making method with Luo Shi Highpure Total Nucleic Acid Kit (pellosil post method), platform is contrast, operate according to specification sheets separately respectively, through the HBV nucleic acid that cracking, cleaning, elution step acquisition are extracted, each about 30min of operation required time.
(3) HBV DNA fluorescence quantitative PCR detection
The HBV DNA that above-mentioned three kinds of methods are extracted detects with HBV nucleic acid fluorescent quantitative PCR test kit, gets test kit HBV PCR damping fluid n * 30 μ l, MgCl
2N * 5 μ l, HBV fluorescent probe n * 5 μ l, Taq enzyme n * 3 μ l mix in a centrifuge tube (n is sample number to be amplified), and the vibration mixing is 10 seconds on the vortex vibrator, and the low-speed centrifugal several seconds is by every pipe 43 μ l packing PCR reaction solutions.Nucleic acid rapid extraction reagent of the present invention is extracted the HBV DNA that obtains, as long as the PCR reaction solution is added above-mentioned containing in the amplification pipe that extracts reagent and blood plasma mixed solution; To pellosil post method, paramagnetic particle method contrast method, the template of then drawing 7 μ l wash-outs adds in the PCR reaction solution amplification pipe of 43 μ l packing.
Carry out augmentation detection at ABI7500 amplified fluorescence instrument, program is: behind 50 ℃ of 2min, the 94 ℃ of 5min according to 93 ℃ of 30 seconds → 60 ℃ 90 seconds cyclic amplifications 40 times.Reaction finishes the back and uses the instrument software analytical results, and three kinds of methods are to 5 routine blood plasma HBV DNA quantitative result such as tables 2, and three kinds of method quantitative results are close as can be known, and the method operation of reagent is easier, the time is shorter, has advantage but employing the present invention extracts.
Table 2 and post method, paramagnetic particle method relatively are used for blood plasma HBV DNA extraction fluorescent PCR detected result