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Detection kit for detecting relative expression level of PML-RAR (alpha) fusion gene by use of fluorescent quantitative PCR (polymerase chain reaction) technology

A detection kit and technology for relative expression, which is applied in the field of detection kits for detecting the relative expression of PML-RARα fusion gene using fluorescent quantitative PCR technology, can solve the problems of high cost and low specificity, and achieve simple operation and high results. Facilitate and improve experimental efficiency

Inactive Publication Date: 2012-10-10
FUZHOU ADICON CLINICAL LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, because SYBR GreenI is an unsaturated dye, its specificity is not as good as that of the double-probe hybridization method and the Taqman method, and its specificity must be judged by observing the melting curve; and the cost of the double-probe hybridization method is relatively expensive.

Method used

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  • Detection kit for detecting relative expression level of PML-RAR (alpha) fusion gene by use of fluorescent quantitative PCR (polymerase chain reaction) technology
  • Detection kit for detecting relative expression level of PML-RAR (alpha) fusion gene by use of fluorescent quantitative PCR (polymerase chain reaction) technology
  • Detection kit for detecting relative expression level of PML-RAR (alpha) fusion gene by use of fluorescent quantitative PCR (polymerase chain reaction) technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The kit of the present invention includes: it includes the following materials in ratio of parts: erythrocyte lysate (50 parts, 50ml, 10×), TRIzol (50 parts, 50ml), chloroform (50 parts, 30ml), absolute ethanol (50 servings, 40ml), reverse transcription PCR reagent (50 servings, 0.8ml), detection system PCR reaction solution (50 servings, 1.15ml), positive control and negative control;

[0026] Among them, 10× red blood cell lysate (NH4CL 82mg / ml, NAHCO3 8.4mg / ml, EDTA-NA2 3.72mg / ml, DEPC-ddH2O to the prepared volume).

[0027] Reagents for reverse transcription PCR include: 5×RT buffer (120μl), RT Enzyme (25μl), Random Primer (30μl).

[0028] The detection system PCR reaction solution includes: Taq polymerase (1μl), dNTPs (4μl), Tris-HCl buffer (12.5μl), magnesium ion (2μl), and the upstream primers for detecting the target gene are: L-F (0.8μl) or S-F ( 0.8μl), the downstream primer is L / S-R (0.8μl) (L-R is the same as S-R, so L / S-R can be expressed as L-R or S-R), t...

Embodiment 2

[0039] The operation flow of this method:

[0040] (1) Extract tissue RNA from blood: Add 1ml of erythrocyte lysate to a clean 1.5ml centrifuge tube, take 0.5ml of anticoagulated blood and mix well. Let stand at room temperature for 10 minutes; centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 0.5ml red blood cell lysate again, centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 1ml TRIzol to the cells, and pipette repeatedly until sedimentation Dissolve completely, let stand at room temperature for 5 minutes; add 0.2ml chloroform, shake evenly; centrifuge at 14000rpm 4°C for 10 minutes, absorb the supernatant layer and transfer to another new centrifuge tube; add an equal volume of isopropanol, mix well up and down, stand at room temperature Centrifuge at 14000rpm at 4°C for 10min, discard the supernatant, add 1ml of 75% ethanol, wash the tube wall upside down gently; centrifuge at 1...

Embodiment 3

[0050] Using the detection kit of the present invention to detect clinical specimens

[0051] A total of 80 cases of anticoagulant blood samples from patients with acute promyelocytic leukemia (APL) submitted for inspection were extracted, and genomic RNA was extracted, reagents were prepared and tested according to the method described in Example 2.

[0052] Take 2 μl of each sample and add it to the detection system PCR reaction solution. At the same time, make a standard curve of positive, negative, blank control, and internal reference gene / target gene. A 96-well fluorescent PCR instrument can detect 38 samples at the same time, each sample is tested twice, a positive control, a negative control and a blank control. The detection time is only 100 minutes.

[0053] The experimental results are compared with the results reported by the special inspection laboratory to determine the accuracy of the sample detection. Some positive results are as follows:

[0054]

[005...

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Abstract

The invention relates to a detection kit for detecting the relative expression level of PML-RAR (alpha) fusion gene by use of fluorescent quantitative PCR (polymerase chain reaction) technology. The detection kit comprises the following materials in part ratio: red blood cell lysis buffer, TRIzol, chloroform, absolute ethyl alcohol, reverse transcription PCR reagent, detection system PCR reaction liquid, positive control substance and negative control substance. The kit provided by the invention has the advantages that the precision is high, the result is convenient to read, both the amplification efficiency and the rate reach the best level and the like; the complicated condition grope link is omitted; and the experimental efficiency is greatly improved. The kit has good specificity and high sensitivity and is easy and convenient to operate according to the test. The detection kit is favorable for the trace residue detection of the PML-PAR (alpha) (type L / type S) fusion gene in a clinical APL (acute promyelocytic leukemia) patient, and has great importance in performing timely intervention therapy to avoid hematological recurrence, adjusting the therapeutic schedule, evaluating the therapeutic effect, predicting the prognosis and preventing clinical recurrence.

Description

Technical field [0001] The invention relates to a detection kit for the relative expression of genes used for clinical testing, which uses Taqman probe real-time fluorescent quantitative PCR technology to detect PML-RARα (L-type) in human acute promyelocytic leukemia (APL) patients. / S type) fusion gene expression level, and at the same time carry out more accurate screening for high-risk groups. The kit can effectively save detection time and improve detection accuracy. technical background [0002] Acute promyelocytic leukemia (APL) is a special type of acute myeloid leukemia, and its specific marker is the translocation of chromosome t(15; 17) (q22; q21), forming PML-RARα fusion gene. More than 85% of APL patients have specific chromosomal translocation t(15; 17)(q22; q21), forming PML-RARα fusion gene. About 95% of the PML-RARα gene exists in the cells, which is an important basis for judging the prognosis and recurrence of APL. Large-scale clinical research shows that...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 方国伟周晓椟王淑一徐建成
Owner FUZHOU ADICON CLINICAL LAB INC
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