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HPV E6E7 mRNA detection test kit based on digital PCR and detection method using test kit

A detection kit and digital technology, applied in the field of HPVE6E7 mRNA detection kits based on digital PCR, can solve the problems of increasing false positive results and background signals, unable to obtain absolute quantitative results, etc., so as to improve the lower limit of detection concentration and improve detection accuracy. the effect of reducing background fluorescence

Inactive Publication Date: 2020-08-25
苏州艾可瑞斯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Because traditional PCR is a large-volume reaction system, non-specific amplification increases false positive results and background signals, so it is ultimately impossible to obtain absolute quantitative results

Method used

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  • HPV E6E7 mRNA detection test kit based on digital PCR and detection method using test kit
  • HPV E6E7 mRNA detection test kit based on digital PCR and detection method using test kit
  • HPV E6E7 mRNA detection test kit based on digital PCR and detection method using test kit

Examples

Experimental program
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Effect test

Embodiment 1

[0093] Use the detection kit of the present invention to detect 4 routine clinical vaginal secretion exfoliated cell specimens, and the detection results are as follows: figure 1 shown. All 4 samples tested positive, sample 1: HPV16, HPV18 E6E7 mRNA double positive ( figure 1 A); Sample 2: HPV18, HPV58 E6E7 mRNA double positive ( figure 1 B); sample 3: positive detection of HPV18, HPV52 E6E7 mRNA ( figure 1 C); Sample 4: HPV16 E6E7 mRNA detection positive ( figure 1 D). Among them, samples 2 and 3 were tested negative by conventional qPCR method.

Embodiment 2

[0095] Five cases of artificially prepared samples with different concentrations were used (artificially prepared samples were extracted from HPV16 positive cell line SiHa and then incorporated into HPV16 negative cell line HaCaT RNA. Gradient HPV16 E6E7mRNA), using the detection method of the present invention and the traditional qPCR method to detect 5 samples respectively, the same concentration and the same method were detected 5 times respectively, and the quantitative results and coefficient of variation of the two methods were compared. The results are as follows: figure 2 shown. The comparison results showed that in the high concentration E6E7 copy template samples, the average coefficient of variation of dPCR was 6% (e.g. figure 2 A). In comparison, qPCR has a coefficient of variation of 13% (eg figure 2 B), the difference is not significant. However, in samples with low concentration of E6E7 copy templates, the average coefficient of variation of dPCR of the pr...

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Abstract

The invention provides an HPV E6E7 mRNA detection test kit based on digital PCR and a detection method using the test kit. The detection test kit comprises the following components: an RNA extractionreagent, a cDNA reverse transcription system and a digital PCR detection system, wherein the RNA extraction reagent comprises the following components: a lysis solution, a deproteinized solution, a rinsing solution, RNA enzyme-free double distilled water and an RNase-Free adsorption column CR3. The cDNA reverse transcription system is prepared from the following components: 10*RT Mix, Super pure dNTPs, oligo-dT, Quant Reverse Transcriptase and RNase-Free water. The digital PCR detection system comprises the following components: a forward primer, a reverse primer, an HPV detection probe, a Genotyping Mastermix, a Droplet Stabilizer and H2O. By adopting the digital PCR technology, the background fluorescence after amplification is greatly reduced, the detection flux is remarkably improved,the lower limit of the detection concentration is improved, the detection accuracy is greatly improved, and absolute quantification can be achieved.

Description

technical field [0001] The invention relates to a detection kit, in particular to a digital PCR-based HPV E6E7 mRNA detection kit and a detection method using the same. Background technique [0002] Because traditional PCR is a large-volume reaction system, non-specific amplification increases false positive results and background signals, so it is ultimately impossible to obtain absolute quantitative results. Digital PCR (dPCR) is a breakthrough nucleic acid quantitative analysis technology that has attracted attention and developed rapidly in recent years. This technology first dilutes the nucleic acid template and distributes it to a large number of independent reaction units, so that each reaction unit has only a single template molecule, and then performs PCR amplification reaction. After the amplification, the fluorescence signal of each reaction chamber is counted. Quantitative analysis of DNA copy number. Digital PCR uses amplification first and then quantification...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851
CPCC12Q1/708C12Q1/6851C12Q2600/166C12Q2563/159C12Q2545/113C12Q2563/107
Inventor 袁嘉扬吴炯戴谦邬升超
Owner 苏州艾可瑞斯生物科技有限公司