Kit for detecting five pathogens of TORCH and application of kit

A pathogen and kit technology, applied in the field of genetic engineering, can solve the problems of poor sensitivity and specificity, long operation time, complex reagents, etc., and achieve the effects of simple operation, good specificity, and accurate and sensitive detection.

Inactive Publication Date: 2020-03-06
廊坊诺道中科医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection of the above five pathogens by immunological methods is not only poor in sensitivity and specificity, but also has a certain window period after infection to the production of IgG or IgM, which may lead to false negatives. Know the test result

Method used

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  • Kit for detecting five pathogens of TORCH and application of kit
  • Kit for detecting five pathogens of TORCH and application of kit
  • Kit for detecting five pathogens of TORCH and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] TORCH detection of five pathogens:

[0040] (1) Extraction of genomic DNA

[0041] To collect the samples of the subject to be tested, a commercially available virus genome DNA / RNA extraction kit was used, and the specific operation process was carried out according to the instructions.

[0042] (2) Primer design

[0043] The corresponding sequences of toxoplasma gondii (TOX), rubella virus (RV), human cytomegalovirus (CMV), herpes simplex virus type I (HSV1) and herpes simplex virus type II (HSV2) were obtained from GenBank, and amplification primers were designed to detect needles to ensure that each pair of primers and probes can amplify the corresponding pathogen and not cause non-specific amplification with other pathogens.

[0044] Table 1 TORCH amplification primers and probes for five pathogens

[0045]

[0046] (3) Establishment of multiplex PCR method

[0047] Using the kit provided in this application, the reaction system is 20uL, specifically: 2uL of nu...

Embodiment 2

[0057] Specificity experiments for primer-probe sets

[0058] Use TORCH five pathogenic plasmids and known pathogen samples to do cross experiments to verify its specificity.

[0059] Add the corresponding primer probes of 5 kinds of TORCH pathogens to the wells of the same row of 96-well plate, and add the positive plasmid templates of the same 5 TORCH pathogens and samples of known pathogens to the wells of the same column. The samples of known pathogens include influenza virus A (FluA), Influenza Virus B (FluB), Coronavirus (OC43), Boca Virus (HBOV), Parainfluenza Virus 1 (HPIV1), Parainfluenza Virus 3 (HPIV3), Rhinovirus (HRV), Pneumonia Mycoplasma (MP) and Chlamydia pneumoniae (CP) ensure that each primer probe has an independent contact opportunity with any pathogen. Through such a cross experiment, the CT values ​​are counted to verify the specificity of the method of the present invention.

[0060] The multiple fluorescent PCR reaction system is 20uL, specifically: 2u...

Embodiment 3

[0064] Sensitivity experiment

[0065] The self-made 5 kinds of TORCH pathogen positive plasmid samples were diluted tenfold, 10 2 -10 5 copy / ul, using the kit provided by this patent to amplify the above-mentioned diluted samples respectively, the detection sensitivity results of the five TORCH pathogens are as follows Figure 1-5 As shown, their R 2 The values ​​are 0.999 (TOX), 0.996 (RV), 0.998 (CMV), 0.998 (HSV1) and 0.991 (HSV2), indicating that the linear relationship is good and the sensitivity can reach 100copies / ul.

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Abstract

The invention relates to the technical field of gene engineering, in particular to a primer probe combination for detecting five pathogens of TORCH, which comprises five pairs of primers and five corresponding probes. The invention further provides a kit for detecting the five pathogens of TORCH. The kit comprises a primer probe combination for detecting the five pathogens of TORCH. The inventionfurther provides an application of the kit for detecting the five pathogens of TORCH in detection or auxiliary detection of non-diagnostic purposes of the five pathogens of TORCH. The kit for detecting the five pathogens of TORCH provided by the invention can be used for simultaneously detecting the five pathogens of TORCH, realizes multiple detection, is high in sensitivity, quick and convenient,and can realize batch detection of samples.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a kit for detecting five pathogens of TORCH and its application. Background technique [0002] TORCH is a group of pathogens that can cause congenital intrauterine infection and perinatal infection, including toxoplasma gondii (TOX), rubella virus (RV), human cytomegalovirus (CMV), herpes simplex virus type 1 (HSV1) and simplex Herpes virus type 2 (HSV2). [0003] After women of childbearing age are infected with one or more pathogens of TORCH, the pathogens will be transmitted vertically to the fetus through the placenta, which can lead to miscarriage, premature birth, stillbirth and deformed children. Fetal malformations caused by Toxoplasma gondii infection include hydrocephalus, cerebellar malformation, chorioretinitis, and brain calcification; rubella virus infection can cause fetal congenital cataract, congenital heart disease, and neurological deafness; cytome...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6893C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6893C12Q1/701C12Q1/705C12Q2600/16C12Q2600/166C12Q2537/143C12Q2563/107C12Q2545/101C12Q2545/113C12Q2521/107
Inventor 孙晋华陈传红胖铁良卢孟孟李建中
Owner 廊坊诺道中科医学检验实验室有限公司
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