Method for quantitative determination of biological safety of nano-material by molecular biology technology
A technology of biological safety and molecular biology, applied in the field of quantitative detection of biological safety of nanomaterials by molecular biology technology, can solve the problems of low spontaneous mutation rate and low background of rpsl analysis, and achieve easy operation and accurate and reliable detection results , the effect of saving workload
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preparation example Construction
[0033] 2. Preparation of nanomaterial suspension solution:
[0034] Commercially available products can be used as the nanomaterial solid powder, which belongs to the prior art and will not be described in detail. Taking the nanomaterial suspension solution with a concentration of 10mg / mL (W / V) as an example, first accurately weigh 10mg of sterilized nanomaterials into a sterilized 1.5mL small centrifuge tube, and slowly Add sterilized water to a volume of 1 mL; then close the cap of the centrifuge tube and place it in an ultrasonic oscillator for ultrasonic treatment for a certain period of time. The specific ultrasonic power and ultrasonic time should be based on the fact that the nanomaterials are completely suspended and can be placed statically at room temperature for at least 10 minutes without occurrence Accumulation and precipitation shall prevail.
[0035] 3. Configuration of in vitro gene amplification system:
[0036] The in vitro gene amplification system is base...
specific example 1
[0048] Specific example 1: Quantitative detection of the mutation rate of PCR to the biosafety of nano Ag particles:
[0049] 1. PCR reaction:
[0050] . Configure the system in a thin-walled PCR tube according to the following order and dosage.
[0051] wxya 2 O 24.4 μL
[0052] 10×PCR buffer (with Mg 2+ ) 5μL
[0053] dNTP substrate (2.5mM) 5μL
[0054] Primer 1 (2.0 μM) 5 μL
[0055] Primer 2 (2.0 μM) 5 μL
[0056] Template (3.75ng / μL) 2.6μL
[0057] Nano Ag particles (0.24μg / μL) 2μL
[0058] Taq enzyme (5U / μL) 0.5μL
[0059] Among them, the template is the linearized plasmid pMOL21, the primer is labeled with biotin at the 5' end, and the Taq enzyme is purchased from Beijing Sanbo Yuanzhi Bioengineering Company.
[0060] The primer sequences are:
[0061] Primer 1: 5′-biotin-AAAAAAAAAAACGCGTCACCAGTCACAGAAAAGCATCTTAC-3′
[0062] Primer 2: 5′-AAAAAAAAAAACGCGTCAACCAAGTCATTCTGAGAATAGT-3′
[0063] 2. Set the cycle conditions on the PCR instrument according to the f...
specific example 2
[0072] Quantitative detection of the mutation rate of isothermal amplification on the biological safety of nano-Au (10nm) colloids:
[0073] 1. Configuration of rolling circle amplification reaction system:
[0074] (1). Sample thermal denaturation and primer and plasmid annealing reaction:
[0075] ddH2O 4.4μl
[0076] 10×buffer 1.0μl
[0077] Random primer (100μM) 2.5μl
[0078] sample (1μg / ml) 1.0μl
[0079] Heat at 95°C for 3 minutes, then place on ice for 15 minutes.
[0080] (2). Amplification reaction: dNTP (10mM) 0.5μl
[0081] 100XBSA 0.1 μl
[0082] Phi29 DNApolymerase (10U / μl) 0.5μl
[0083] Nano-Au (10 nm) colloid was added to make the final reaction system 20 μl, and incubated overnight at 30°C.
[0084] (3). Heat inactivation of phi29 DNA polymerase: 65°C, 10 minutes.
[0085] 2. After purification, the amplified product was digested with 10 units of EcoR I at 37°C for more than 6 hours, and then purified.
[0086] 3. Carry out the fifth step in the abo...
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