Fluorescent quantitative PCR kit for ovarian cancer, detection method and application
A fluorescence quantification and kit technology, applied in the field of medical testing, can solve the problems of complex operation, low sensitivity, limited specificity and sensitivity, etc., and achieve the effect of easy collection, high sensitivity, simple and fast method
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Embodiment 1
[0080] 1. Reagent kit
[0081] Embodiment 1 provides a kind of fluorescent quantitative PCR detection kit for ovarian cancer, such as figure 1 As shown, the kit includes a box body 1 and reagent tubes for containing reaction reagents, the box body 1 is divided into a first area 11, a second area 12 and a third area 13, so that different volume sizes The reagent tubes are stored respectively, wherein the first area 11 and the second area 12 respectively account for 1 / 5 of the volume of the box body, and the third area 13 accounts for 3 / 5 of the volume of the box body; Area 11 includes a standard tube 4, the second area 12 includes a primer tube 5, and the third area 13 includes an RT-PCR reaction solution tube 2 for ovarian cancer-specific mRNA reverse transcription, a fluorescent quantitative PCR reaction Liquid line 3 and nuclease-free water line 6.
[0082] Among them, the standard quality tube is composed of a plasmid standard product tube containing ETV4, a plasmid stand...
Embodiment 2
[0119] The difference between embodiment two and embodiment one is:
[0120] The standard tube in Example 2 is only the plasmid standard tube containing FOXM1, which contains 10 μl of the standard plasmid containing the gene to be tested FOXM1 at a concentration of 50 ng / μl.
[0121] The primer tube is a FOXM1-specific primer tube, containing 100 μl of specific primers for amplifying the gene to be tested FOXM1, wherein the concentrations of primer 3 and primer 4 for FOXM1-specific amplification are 0.8 μM, respectively.
[0122] Among them, the volume of the RT-PCR reaction liquid tube is 2ml, and it is equipped with the reverse transcription reaction system of RT-PCR master mix (RT-PCR Master Mix), which consists of RTase M-MLV, M-MLV Buffer, oligo(dT) primer (oligo(dT)primer), nuclease inhibitor (RNase inhibitor) and dNTPs, 1ml in total, the concentration of RTase M-MLV is 30U / μl, the concentration of oligo(dT)primer is 60μM, and the concentration of RNase inhibitor is 3U / ...
Embodiment 3
[0135] The difference between embodiment three and embodiment one is:
[0136] The standard tube in Example 3 is only the plasmid standard tube containing ETV4, which contains 10 μl of the standard plasmid containing the gene ETV4 to be tested at a concentration of 50 ng / μl.
[0137] The primer tube is an ETV4-specific primer tube, which contains 100 μl of specific primers for amplifying the gene ETV4 to be tested. Wherein, the concentrations of primer 1 and primer 2 used for ETV4-specific amplification were 1 μM, respectively.
[0138] Among them, the volume of the RT-PCR reaction liquid tube is 2ml, and it is equipped with the reverse transcription reaction system of RT-PCR master mix (RT-PCR Master Mix), which consists of RTase M-MLV, M-MLV Buffer, oligo(dT) primer (oligo(dT)primer), nuclease inhibitor (RNase inhibitor) and dNTPs, 1ml in total, the concentration of RTase M-MLV is 80U / μl, the concentration of oligo(dT)primer is 80μM, and the concentration of RNase inhibitor...
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