PCA3 gene detection method and primer in human urine
A urine, amplification primer technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc. High repetition stability and damage reduction effect
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Embodiment 1
[0031] A method for detecting PCA3 gene in human urine:
[0032] 1. Collection and processing of urine samples
[0033] The traditional prostate massage method is used, that is, the examiner makes a digital rectal examination, touches the prostate on the front wall of the rectum, massages symmetrically from the left and right sides to the central groove three times, and then massages the central groove from the bottom of the prostate to the tip three times, and then instructs the patient to urinate and collect Initial urine 50ml, immediately put into cold water to cool. Centrifuge at 2500r / min at 4°C for 5 minutes, collect the urine sediment, and add an appropriate amount of pre-cooled PBS washing solution twice. Collect the urine sediment in a 1.5ml centrifuge tube, add Trizol reagent and repeatedly blow and mix, and put it in a -80°C refrigerator for later use.
[0034] 2. Total RNA Extraction
[0035] Thaw the above urine sediment sample added with Trizonl at 4°C.
[00...
Embodiment 2
[0067] A kit for the diagnosis of prostate cancer, comprising:
[0068] (1) Collect urine sediment reagents and utensils containing prostate cells, including: digital rectal examination urine collection tube, PBS washing solution, 1.5ml centrifuge tube, Trizol reagent;
[0069] (2) Reagents for extracting total RNA in urine, including: chloroform, isopropanol, 75% ethanol, absolute ethanol, DEPC water;
[0070] (3) Reagents for detecting the expression of PCA3 and PSA genes, including reverse transcription reaction solution and fluorescent PCR reaction solution. Wherein the reverse transcription reaction solution includes 5X Buffer, reverse transcriptase, dNTP and DEPC water; the fluorescent PCR reaction solution includes 5X Buffer, dNTP, TaqMen enzyme, primers and DEPC water as shown in SEQ ID NO:3-8;
[0071] (4) Calculation formula of urine PCA3 score in the sample: PCA3 score=(PCA3 mRNA / PSA mRNA)×1000.
Embodiment 3
[0073] Utilize the test kit of embodiment 2 to detect clinical sample:
[0074] The initial urine after prostate massage was collected from male patients sent to urology department. Among them, 37 cases of prostate cancer, aged 39-78 years, with an average age of 70.6 years; 68 cases of benign prostatic hyperplasia, aged 59-87 years, with an average age of 68.2 years; Patients with urinary calculus of prostate disease were used as normal controls, aged 30-43 years old, with an average of 36.3 years old.
[0075] Urine total RNA was extracted according to the method described in Example 1, and the urine PCA3 score was detected by fluorescent PCR method. Each sample was repeated 3 times, and positive, negative, and blank controls were made at the same time. Judgment based on scoring results.
[0076] The PCA3 score of the control group was 13.5﹢8.93, that of the benign prostatic hyperplasia group was 30.56﹢38.91, and that of the prostate cancer group was 158.89﹢118.25. There ...
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