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Mixed gene, standard plasmid and kit for detecting fusion gene as well as preparation method of standard plasmid

A technology of fusing genes and standard plasmids, applied in the field of molecular biology, can solve the problems of difficulty in guaranteeing the accuracy and consistency of detection results, small detection range, complicated operation, etc., to improve detection efficiency and accuracy, reduce detection costs, Simple to use effects

Active Publication Date: 2018-10-19
上海科医联创医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Single fluorescence standard curve, there is an ideal state that requires gene amplification efficiency to reach 100%, which is difficult to achieve in the actual detection process, and the accuracy and consistency of the detection results are difficult to guarantee
In addition, due to the current quantitative items of blood disease fusion genes, the double standard curve method requires the synthesis of multiple target gene plasmids, which has the disadvantages of complicated operation, easy contamination, and small detection range.

Method used

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  • Mixed gene, standard plasmid and kit for detecting fusion gene as well as preparation method of standard plasmid
  • Mixed gene, standard plasmid and kit for detecting fusion gene as well as preparation method of standard plasmid
  • Mixed gene, standard plasmid and kit for detecting fusion gene as well as preparation method of standard plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] This example is the mixed gene, standard plasmid and preparation method used in the present invention.

[0058] The mixed gene for detecting fusion gene of the present invention, its gene sequence is as shown in SEQ ID NO: 1, and it is synthesized by gene by BCR-ABL fusion gene, AML-ETO fusion gene, PML-RARA fusion gene and ABL internal reference gene way to get.

[0059] Wherein, the BCR-ABL fusion gene is shown in SEQ ID NO: 2, the AML-ETO fusion gene is shown in SEQ ID NO: 3, the PML-RARA fusion gene is shown in SEQ ID NO: 4, and the ABL internal reference gene is shown in SEQ ID NO:5 shown.

[0060] Insert the above-mentioned mixed gene into a plasmid vector according to certain requirements to obtain a standard plasmid.

[0061] Wherein the plasmid vector is a PCU57 plasmid, the gene sequence of which is shown in SEQ ID NO:6.

[0062] The preparation method of the standard plasmid for detecting fusion gene of the present invention is as follows, comprising:

[...

Embodiment 2

[0075] This example is to carry out the linearity verification and sensitivity verification of the ABL internal reference gene on the standard plasmid prepared in Example 1.

[0076] The specific steps of verification are as follows:

[0077] Step S51, measure the standard plasmid to obtain the concentration, absorbance and copy number of the standard plasmid.

[0078] The detection instrument is nanodrop2000, the absorbance is A260 absorbance, and the calculation formula of the copy number is shown in formula (1).

[0079] Copy number=50*A260 absorbance*6.02*10 14 / ((plasmid sequence length+mixed gene sequence length)*660)(1)

[0080] Step S52, performing gradient dilution of the standard plasmids to obtain standard plasmids with different copy numbers.

[0081] Among them, the copy numbers after serial dilution are 1*10 0 pcs / ml, 1*10 1 pcs / ml, 1*10 2 pcs / ml, 1*10 3 pcs / ml, 1*10 4 pcs / ml, 1*10 5 pcs / ml, 1*10 6 pcs / ml, 1*10 7 pcs / ml, 1*10 8 pcs / ml, 1*10 9 pieces / ...

Embodiment 3

[0089] This example is to perform linearity verification and sensitivity verification of the BCR-ABL fusion gene on the standard plasmid prepared in Example 1.

[0090] The specific steps of verification are as follows:

[0091] Step S51, measure the standard plasmid to obtain the concentration, absorbance and copy number of the standard plasmid.

[0092] The detection instrument is nanodrop2000, the absorbance is A260 absorbance, and the calculation formula of the copy number is shown in formula (1).

[0093] Copy number=50*A260 absorbance*6.02*10 14 / ((plasmid sequence length+mixed gene sequence length)*660)(1)

[0094] Step S52, performing gradient dilution of the standard plasmids to obtain standard plasmids with different copy numbers.

[0095] Among them, the copy numbers after serial dilution are 1*10 0 pcs / ml, 1*10 1 pcs / ml, 1*10 2 pcs / ml, 1*10 3 pcs / ml, 1*10 4 pcs / ml, 1*10 5 pcs / ml, 1*10 6 pcs / ml, 1*10 7 pcs / ml, 1*10 8 pcs / ml, 1*10 9 pieces / ml.

[0096] ...

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Abstract

The invention relates to a mixed gene for a detecting fusion gene. A gene sequence of the mixed gene is shown in SEQ ID NO:1. The mixed gene comprises a BCR-ABL fusion gene shown in SEQ ID NO:2, an AML-ETO fusion gene shown in SEQ ID NO:3, a PML-RARA fusion gene shown in SEQ ID NO:4 and an ABL reference gene shown in SEQ ID NO: 5. The invention further relates to standard plasmid containing the mixed gene, and the standard plasmid comprises PUC57 plasmid shown in SEQ ID NO: 6. The invention further relates to a kit containing the standard plasmid and further relates to a preparation method ofthe standard plasmid. The mixed gene has the advantages that the mixed gene can be applied to establishing a double-standard curve which is used for quantitatively detecting BCR-ABL, RUNX1-RUNX1T1 (AML-ETO) and PML-RARA fusion gene in a leukemia related fusion gene in human marrow or a peripheral blood sample; detection efficiency and accuracy are effectively improved, manual operation errors arereduced, and multi-plasmid pollution is prevented from happening; the lowest detection copy number of the standard plasmid is 1*10<0> per ml; operation is simple, pollution is not prone to happening,the detection range is large, and detection cost can be effectively reduced.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a mixed gene for detecting a fusion gene, a standard plasmid, a kit and a preparation method thereof. Background technique [0002] At present, for the quantitative detection of clinical blood disease fusion genes, the synthetic ABL internal reference gene is mainly used as a single fluorescence standard curve for relative quantification; there is also a dual fluorescence quantitative standard curve for synthetic ABL internal reference gene and target gene sequence for quantification. [0003] The single fluorescence standard curve requires the ideal state of gene amplification efficiency to reach 100%, which is difficult to achieve in the actual detection process, and the accuracy and consistency of the detection results are difficult to guarantee. In addition, due to the current quantitative items of blood disease fusion genes, the double standard curve method require...

Claims

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Application Information

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IPC IPC(8): C12Q1/6811C12N15/11
CPCC12Q1/6811C12Q2545/101C12Q2545/113C12Q2563/107
Inventor 曹金良
Owner 上海科医联创医学检验所有限公司
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