Primer probe combination, kit and method for quantitatively detecting JAK2 V617F gene mutation rate

By using a specific primer-probe combination and the 2-ΔΔCt method of the internal reference gene GPI, combined with mismatched bases and hairpin structures, the sensitivity and accuracy problems of JAK2 V617F mutation detection in the existing technology were solved, and efficient and economical quantitative detection of JAK2 V617F mutation rate was achieved.

CN120738338APending Publication Date: 2025-10-03HENAN PROVINCE HOSPITAL OF TCM THE SECOND AFFILIATED HOSPITAL OF HENAN UNIV OF TCM
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Patent Information

Application Number
CN202510859399.7
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2025-06-25
Publication Date
2025-10-03

AI Technical Summary

Technical Problem

Existing technologies lack sensitivity in detecting JAK2 V617F gene mutations, making it difficult to meet the needs of low-abundance mutations or dynamic monitoring. In addition, the operation is complex and costly, making it difficult to achieve high-sensitivity and high-precision quantitative detection.

Method used

A specific primer-probe combination was used in combination with the internal reference gene GPI to quantitatively calculate the JAK2 V617F mutation rate using the 2-ΔΔCt method. Mismatched bases were introduced to inhibit nonspecific amplification, and hairpin oligonucleotides were used to inhibit nonspecific reactions, achieving closed-tube operation.

Benefits of technology

It achieves high sensitivity (minimum detection limit 0.1%), high specificity and high efficiency in JAK2 V617F mutation rate detection, simplifies result interpretation, avoids product contamination, and reduces operational complexity and cost.

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Abstract

The invention provides a primer and probe combination, a kit and a method for quantitatively detecting the JAK2V617F gene mutation rate, and the primer and probe combination comprises a primer and probe set targeting JAK2V617F mutation sites and a primer and probe set of reference genes GPI, according to the primer probe set targeting JAK2V617F mutation sites, the nucleotide sequence of an upstream primer of a specific mutation site is any one of SEQ ID NO.1-7, the nucleotide sequence of an upstream primer of a specific wild type site is any one of SEQ ID NO.9-14, the nucleotide sequence of a universal downstream primer is SEQ ID NO.8, and the nucleotide sequence of a probe is SEQ ID NO.15. The invention further discloses a kit for detecting the JAK2V617F mutation sites. According to the invention, the JAK2V617F mutation rate can be economically, simply, efficiently, sensitively, accurately and quantitatively detected, and the detection mutation rate is as low as 0.1%.
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