A Direct Real-time Fluorescent Quantitative PCR Method
A real-time fluorescence quantitative and direct technology, applied in the fields of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of prolonging the detection time, increasing the experimental operation steps, etc., to achieve the effect of easy operation
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Embodiment 1
[0037] Polystyrene microsphere-coated PCR amplification reagent part:
[0038] 1. Coating of polystyrene microspheres:
[0039] Take 10 polystyrene microspheres with a diameter of 1-2mm, and coat them on the bottom of the PCR amplification tube with 10 μl of paraffin oil mixture with a melting point of 58°C-60°C through melting and condensation.
[0040] 2. Preparation of fluorescent PCR amplification reagents:
[0041] 20 μl of fluorescent PCR amplification reagent contains: 1 μl of 1.5 U / μl hot-start Taq DNA polymerase; 1 μl of 20 μmol / L upstream primer, 1 μl of 20 μmol / L downstream primer and 1 μl of 10 μmol / L probe ; 4 μl of 25mmol / L magnesium chloride; 2 μl of 500mmol / L potassium chloride; 1 μl of four mixed dNTPs (each 10mmol / L); 9 μl of acetic acid buffer (PH value 4.0), the above components were mixed Prepare fluorescent PCR amplification reagents. The preparation method of the fluorescent PCR amplification reagent is the same as the preparation method of the PCR am...
Embodiment 2
[0046] Embodiment 2: Sensitivity test of the method for direct fluorescence quantitative PCR
[0047] The clinical HBV DNA quantitative detection value was 1×10 9 For the serum of patients with hepatitis B in IU / ml, the samples that were detected as negative by HBV DNA were used as the diluent, and then sequentially diluted 10 times to obtain the HBV DNA concentration from 1×10 9 IU / ML~1×10 2 IU / ml of sample.
[0048] 1) Add 5 μl of sample into the PCR tube prepared in Example 1, and gently shake the PCR amplification tube to mix the nucleic acid lysate in the tube with the added serum to be tested. The liquid was then thrown to the bottom by centrifugation at 200g / min.
[0049] 2) Place the PCR tube in the PCR instrument for amplification;
[0050] The PCR amplification program was: 56°C, 5 minutes; 95°C, 10 minutes; 50 cycles of 95°C, 10 seconds and 61°C, 30 seconds PCR amplification. Fluorescence signal was detected at 61°C.
[0051] The commercial kit was purchased f...
Embodiment 3
[0053] Embodiment 3: Repeatability test and coefficient of variation analysis of the method for direct fluorescence quantitative PCR
[0054] The experimental method is the same as in Example 2, except that only the preparation of HBV DNA concentration is 5 × 10 3 IU / ml, 5×10 6 IU / ml and 5×10 8 IU / ml of sample is used for repeatability. The patented method was used to detect the above samples in 5 multiple wells respectively. At the same time, a commercial kit was used to conduct a comparative test using the same scheme. From attached image 3 and 4 It can be seen from the data in Table 1 that the repeatability of the patented method is significantly better than that of commercial reagents, and its CV value is 3-11 times lower than that of commercial reagents.
[0055] Table 1: Repeatability test and coefficient of variation analysis data
[0056]
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