Solid-phase multiplex row PCR detection kit and detection method for infectious eye disease pathogens

A technology for detecting kits and pathogens, which is applied in the field of solid-phase multiplexed PCR detection kits for infectious eye disease pathogens, can solve the problem of inability to provide a variety of virus screening tests, affecting specificity and amplification efficiency, and being unfavorable for promotion, etc. problems, to achieve the effect of shortening preparation time, reducing primer dimers, and saving detection time

Active Publication Date: 2020-12-15
海南省百维恩生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Real-time quantitative PCR can quantify the starting template, but the disadvantage is that only one virus can be detected at a time, such as screening and detection of 8 kinds of viruses at the same time, this technology is costly, time-consuming, and labor-intensive; The amount of specimens collected from the eyes is too small to provide screening tests for multiple viruses
Multiplex PCR...

Method used

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  • Solid-phase multiplex row PCR detection kit and detection method for infectious eye disease pathogens
  • Solid-phase multiplex row PCR detection kit and detection method for infectious eye disease pathogens
  • Solid-phase multiplex row PCR detection kit and detection method for infectious eye disease pathogens

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Experimental program
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Effect test

Embodiment example 1

[0083] Implementation Case 1. Design combination of primers and probes that can preferably simultaneously detect 8 human herpes viruses

[0084] The combination of primers and probes designed in the present invention is based on fluorescent quantitative PCR technology, using the combination of several different fluorescent groups, combined with the ability of the instrument to detect fluorescence of different channels to realize real-time quantitative detection of multiple targets.

[0085] Put 2-3 pairs of different amplification primers and corresponding Tagman probes (6FAM, HEX, CY5) of different wavelengths in the same PCR tube to realize the simultaneous detection of 2-3 viral targets. For 8 kinds of herpes viruses, 3 PCR tubes are used, and each tube has 2 to 3 pairs of different primers and corresponding probes, so that the simultaneous screening and detection of 8 kinds of herpes viruses can be realized.

[0086] The primers and probes were designed based on the genome...

Embodiment example 2

[0089] Implementation Case 2. Primer and Probe Solid Phase Technology

[0090] Eight herpesvirus-labeled probes are: 6FAM fluorescent probe (HSV1, EBV); Cy5 fluorescent probe (HSV2, CMV, HHV6); HEX fluorescent probe (VZV, HHV7, HHV8).

[0091] Primers and probes are immobilized using a dry method to immobilize primers and probes to the inner wall of the PCR tube. 8 kinds of herpes virus primers and corresponding probes were divided into three strips of PCR tubes, and the herpes viruses corresponding to the primers and probes in each tube were: the first tube was HSV1, VZV and CMV; the second tube was HSV2 and HHV8; 3 tubes for EBV, HHV6 and HHV7, such as figure 1 ;

[0092] The specific implementation steps of solid-phase treatment are as follows:

[0093] (1) After dissolving primers and probes in RNase-free water, if figure 1 As shown, the upstream and downstream primers and probes were dispensed into corresponding PCR tubes according to the concentration required for th...

Embodiment example 38

[0099] Implementation case 3. Eight kinds of herpes virus solid phase serial multiplex PCR detection kits

[0100] The components of the PCR kit are shown in Table 3.

[0101] Table 3. Components of 8 kinds of herpesvirus solid-phase serial multiplex PCR detection kits

[0102]

[0103]

[0104] The reaction system of the kit is shown in Table 4.

[0105] Table 4. Solid phase serial multiplex PCR reaction system

[0106] 2×PCR Mix 10 μL immobilized primer probe solid state Tear fluid nucleic acid sample / positive control / negative control 2μL RNase-free water 8μL total 20 μL

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Abstract

The invention discloses a solid-phase multiplex row PCR detection kit and a detection method for infectious eye disease pathogens. The solid-phase multiplex row PCR detection kit comprises a PCR mixedsolution, immobilized primer and probes, a positive control, a negative control and RNase-free water, wherein the immobilized primers and probes are eight herpes virus primers and probes subjected toimmobilization treatment and are separately arranged in triple row PCR tubes, and 2-3 herpes virus primers and different fluorescently labeled probes are arranged in each PCR tube to detect eight herpes viruses simultaneously. The scheme of the invention has high sensitivity and high specificity, primer dimers can be reduced, and the amplification efficiency can be improved; by collecting tear, cornea infected parts, tissues, aqueous humor or vitreous humor and other specimens from the eyes of a patient for detection, disease-associated viruses can be simply, quickly and accurately screened to form etiological diagnosis, and clinical targeted medication can be assisted.

Description

technical field [0001] The invention belongs to the technical field of virus detection and relates to a PCR detection kit, in particular to a solid-phase multiplex PCR detection kit and a detection method for infectious eye disease pathogens. Background technique [0002] Viruses, a large group of pathogens, are the most common cause of infectious eye diseases and can cause viral keratitis (epithelitis, stroma, endotheliitis, trabeculitis), viral uveitis and viral retina Inflammation and so on. Clinically, the incidence of viral infectious eye diseases is much higher than that caused by bacteria, fungi or other pathogens. Globally, the incidence and blindness of viral infectious eye diseases are high. For viral keratitis alone, the incidence rate reaches 150 / 100,000[1,2], and there are 10-100,000 new cases every year. 30[3]. Recurrent viral keratitis can lead to severe corneal scarring and even corneal perforation; according to the WHO bulletin, corneal scarring caused by...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/705C12Q1/686C12Q2600/16C12Q2537/143C12Q2563/107C12Q2561/101C12Q2565/501
Inventor 姚玉峰许叶圣郑利斌
Owner 海南省百维恩生物科技有限责任公司
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