Diagnosis reagent for detecting survivin expression in tested specimen, method for detection and use thereof
A diagnostic reagent, survivin technology, applied in the direction of biochemical equipment and methods, microbial determination/testing, etc.
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Embodiment 1
[0074] RT-PCR-ELISA method to detect the expression of survivin
[0075] Materials and methods
[0076] RT-PCR
[0077] The MCF-7 breast cancer cell line was purchased from ATCC and cultured in 10% FCS medium containing 1% penicillin and streptomycin, according to the ATCC manual, at 37°C, 95% air and 5% CO 2 cultivated under conditions. After confluence, the cells were harvested, counted, and stored at -80°C for later use.
[0078] Total RNA of cultured cells was extracted with Trizol reagent (Gibco), and cDNA was synthesized with RT kit (Gibco) according to the manufacturer's instructions.
[0079] The PCR amplification of survivin transcripts used the primers of SEQ ID NO.1 and SEQ ID NO.2, and the cycle parameters were: denaturation at 94°C for 1 minute, annealing at 62°C for 1 minute, extension at 72°C for 2 minutes, cycled 30 times, and finally Incubate at 72°C for 10 minutes. The amplified 437bp survivin DNA was separated by 2% agarose gel electrophoresis, and the ...
Embodiment 2
[0089] Comparison of RT-PCR and RT-PCR-ELISA results
[0090] Materials and methods
[0091] 34 blood samples came from 18 healthy controls and 16 cancer patients. After centrifugation, the total RNA in the cell pellet was extracted with Trizol reagent (Gibco), and cDNA was synthesized with a reverse transcription kit (Gibco). Carry out PCR amplification part cDNA (2 μ l) with fluorescein-labeled SEQ ID NO.5 and SEQ ID NO.6 primers by the condition of embodiment 1, measure amplified with the hybridization method of ELISA formula described in embodiment 1 Amount of survivin cDNA.
[0092] The expression of survivin mRNA was also calculated by the ratio of the amplified survivin cDNA to the amplified β-actin cDNA, which was amplified using a pair of primers specific to the human β-actin gene (5'-GTG GGG CGC CCC AGG CAC CV-3'), 144-163 bases of human actin gene; 5'-CTC CTT AAT GTC ACG CAC GAT TT-3', 660-682), PCR for amplifying human β-actin gene 30 cycles, each cycle includin...
Embodiment 3
[0098] Clinical Application Trial of Survivin RT-PCR-ELISA in China
[0099] Materials and Methods
[0100]As a control, 537 blood samples came from 315 non-cancer individuals, and 222 blood samples from various cancer patients came from three hospitals in Chengdu, Sichuan, China. Cancer types include: lung cancer (65 cases), breast cancer (13), lymphoma (12) , 86 cases of ovarian cancer (20), gastrointestinal cancer (23), rectal cancer (13), liver cancer (6), kidney cancer (8) and other cancers, total RNA extraction, cDNA synthesis, PCR amplification and ELISA The hybridization was carried out as described in Example 1 and Example 2.
[0101] Statistical analysis of data was performed using Mann Whitney rank sum test and ANOVA onRanks and Chi-square Test. For all tests, P<0.05 was considered statistically significant.
[0102] result
[0103] As shown in Figure 3, in the control group, the amplified amount of survivin mRNA was not normally distributed, the median value was...
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