PCA3 and PSA RNA detection kit and amplification system

Abstract

The invention relates to a PCA3 and PSA RNA detection kit and amplification system. The detection kit includes primers and fluorescent probes aiming at PCA3 and PSA genes, and a Stoffel fragment and Tfl DNA polymerase; the primers aiming at the PCA3 gene are shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the sequence of the fluorescent probe is shown as SEQ ID NO. 3; and the primers aiming at the PSAgene are shown as SEQ ID NO. 10 and SEQ ID NO. 11, and the sequence of the fluorescent probe is shown as SEQ ID NO. 12. Based on existing kits, the detection and amplification system of real-time fluorescence quantitative RT-PCR can be optimized; the PCA3 gene selects the primers and probes which have better detection effects and are designed by the sequence on exon 3; optimized Tris-MOPS-sodiumcitrate buffer which is more suitable for RNA amplification can be introduced into a reaction system, and the Stoffel fragment or the Tfl DNA polymerase is introduced as well; and therefore, the kit can tolerate more templates while detection sensitivity and specificity are enhanced.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a real-time fluorescent quantitative RT-PCR detection kit and an amplification system for PCA3 and PSA RNA in urine exosomes. Background technique [0002] Prostate cancer is one of the most common male malignant tumors in developed countries in Europe and the United States. According to data from the American Cancer Society in 2018, the incidence of prostate cancer ranks first in the incidence of male tumors in the United States, and the death rate ranks second. In my country, with the continuous improvement of living standards and environmental changes, the incidence of prostate cancer is also increasing year by year. About 60% of prostate cancer patients in our country are patients with advanced metastases when they are first diagnosed, which will have a direct impact on the treatment effect and long-term survival of prostate cancer patients in our countr...

AI Technical Summary

Problems solved by technology

Prostate biopsy and treatment also have risks: 0.6% to 4.1% of infectious complications from biopsy, and an estimated 20% to 70% of sexual function problems and 5% to 50% of urinary problems from radical prostate surgery

The real-time fluorescence quantitative RT-PCR kit of PCA3 gene and PSA gene RNA originally developed by the applicant has good sensitivity, high specificity and repeat stability. However, in practical application, it is found that the sensitivity is still very unstable. This may be related to the majority of protein in urine and the instability of the amount of target nucleic acid in exosomes. The applicant has improved the detection method by introducing Stoffel fragment or Tfl DNA polymerase, and at the same time introduced a more suitable for RNA amplification. Optimized Tris-MOPS-sodium citrate buffer

Subsequent sample testing found that while the sensitivity increased, the specificity was unstable

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