Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit and detection method of HBV (Hepatitis B Virus) and TP (Treponema Pallidum) of donor corneas

A detection kit and fluorescence quantitative technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problems of low sensitivity, PCR false positive pollution, false negative, etc., and achieve quantitative accuracy High, to avoid the effect of cross-contamination

Active Publication Date: 2012-07-04
SHANDONG EYE INST
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The serum immunology method can be completed by the laboratory. This method is cheap and easy to operate, but has low sensitivity and is prone to false negatives. It can be used as a primary screening method
Moreover, this method cannot be implemented when donor blood samples cannot be obtained; genetic testing can be divided into qualitative testing and quantitative testing, and the current research development trend is the rapid development of new technologies that can be quickly and quantitatively tested with low cost and easy to promote
Although the polymerase chain reaction technology has been widely used in nucleic acid analysis, the false positive pollution and quantitative accuracy of ordinary PCR have always troubled people.
It relies on the post-processing of various types of PCR, and these processing processes can easily cause a large number of PCR products to fly into the air, making the false positive contamination of PCR the biggest problem in large-scale clinical applications
On the other hand, the quantification of all these methods is carried out for the final product of PCR, and the platform effect of PCR greatly interferes with the correlation between the number of original templates of PCR and the final product, making it difficult to improve the quantitative accuracy

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit and detection method of HBV (Hepatitis B Virus) and TP (Treponema Pallidum) of donor corneas

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Detection of HBV virus in donor cornea:

[0047] 1. Extraction of donor eyeball conjunctival tissue DNA and construction of positive working standards:

[0048] (1) Take 5 mg of the conjunctival tissue of the donor eyeball, put the tissue block into a centrifuge tube and chop it up, and add 180 μL of tissue lysis solution. Add 20 μL proteinase K, mix well, and incubate at 56°C until the tissue is completely lysed;

[0049] (2) Add 200 μL of purified lysate, mix with a vortex shaker for 15s, and incubate at 70°C for 10min. Add 200 μL of absolute ethanol to the sample, and mix with a vortex shaker for 15s;

[0050] (3) Transfer the sample from step 2 to the filter column, close the lid tightly and centrifuge at 8000 rpm for 1 min. Discard the waste liquid in the filter column, add 500 μL of eluent 1, and then centrifuge at 8000 rpm for 1 min;

[0051] (4) Discard the waste liquid in the filter column, add 500 μL of eluent 2, and centrifuge at 12000 rpm for 3...

Embodiment 2

[0066] Example 2 Donor corneal TP detection:

[0067] 1. Extraction of donor eyeball conjunctival tissue DNA and construction of positive working standard: The specific steps are the same as those of donor eyeball conjunctival tissue DNA extraction in Example 1.

[0068] A plasmid containing the TP gene (pUC57-TP) was constructed as a positive working standard. The positive working standard contains the target sequence amplified by the TP forward and reverse primers. In addition, an additional sequence is added on each side of the target sequence to make it closer to the structure of the actual test sample, so as to ensure the consistency with the actual test sample. Consistency of amplification efficiency.

[0069] Use Promega PureYield plasmid miniprep system kit to extract TP positive working standard plasmid, and then use UV spectrophotometer at 260nm and 280nm colorimetric to obtain the purity and concentration of TP positive working standard. According to copy number=(...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a fluorescent quantitative PCR detection kit and a detection method of HBV and TP of donor corneas. The kit comprises a DNA (deoxyribonucleic acid) extraction reagent, a DNA amplification reagent and positive working standards, wherein the DNA amplification reagent comprises a premix reagent, a 20X fluorescent probe reinforcing agent, forward and reverse HBV primers, an HBV fluorescent probe, forward and reverse TP primers, a TP fluorescent probe, distilled water of ribose-free nuclease and HBV and TP negative control and positive control; the positive working standards are pUC57-HBV plasmids containing destination sequences amplified by the forward and reverse HBV primers and a pUC57-TP plasmids containing destination sequences amplified by the forward and reverse TP primers. The invention realizes the complete closed-tube detection on donor conjunctiva tissue specimens and does not need PCR aftertreatment, and thereby, cross contamination is avoided; meanwhile, real-time detection is adopted, and an obtained Ct value and the original template number are completely in linear correlation; the quantitative accuracy rate is extremely high, and the relative error is about 50 percent, and thereby, the invention is enough to adapt to the demands of clinical nucleic acid quantification.

Description

technical field [0001] The invention belongs to the field of molecular biology technology detection of pathogenic infection of donor cornea, and particularly relates to a rapid detection kit and detection method for hepatitis B virus (HBV) and Treponema pallidum (TP), major pathogenic pathogens of donor cornea. Background technique [0002] The biosafety of eye banks in my country is extremely poor, and the problem of iatrogenic infection of the donor cornea is not controlled. Because the information on the general condition of eye bank donors is incomplete, the donor who died unexpectedly does not even have medical information on the general condition, and there is a great risk of infection with various pathogens. Therefore, when the donor cornea is very scarce, it is necessary to Under the premise of ensuring safety, the scope of clinical use of the donor cornea should be maximized. At present, the main problem of my country's eye bank is how to establish a method to use ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04G01N21/64
Inventor 谢立信陈鹏
Owner SHANDONG EYE INST
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products