A PCR primer combination, kit and application for the diagnosis of tuberculous ulcer based on rt-qPCR

A diagnostic kit and primer combination technology, applied in the field of molecular biology, can solve the problems of serological detection specificity, low sensitivity, low positive rate of pus acid-fast bacillus culture, inconvenience to repeatedly sample materials, etc., and achieve timely detection methods. , good amplification efficiency, high stability effect

Active Publication Date: 2021-02-19
南京市中西医结合医院
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Problems solved by technology

However, in practice, the positive rate of acid-fast bacilli culture in pus is extremely low and the culture period is long; wound histopathological examination is an invasive examination, which is prone to false negatives and is not convenient for repeated sampling; and serological detection specificity, Sensitivity is not high
Since the pathogenesis of tuberculous ulcer is not yet clear and clinical manifestations are very similar to some chronic refractory wounds, there is no detection index and detection method with high specificity, sensitivity, quickness and convenience for the diagnosis of tuberculous ulcer.

Method used

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  • A PCR primer combination, kit and application for the diagnosis of tuberculous ulcer based on rt-qPCR
  • A PCR primer combination, kit and application for the diagnosis of tuberculous ulcer based on rt-qPCR
  • A PCR primer combination, kit and application for the diagnosis of tuberculous ulcer based on rt-qPCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Sample Collection

[0030] Tuberculous ulcer group (CTB): Whole blood samples were collected from 108 patients with tuberculous ulcer who were pathologically diagnosed as tuberculous ulcer in Nanjing Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Nanjing University of Traditional Chinese Medicine from 2018 to 2019, including 41 males and 67 females. , the age of the patients was 18-80 years old, with an average of 35.4±2.85 years old;

[0031] Control group (CG): Whole blood samples were collected from 36 healthy people with no systemic diseases in the systemic examination, including 14 males and 22 females, aged 18-78 years, with an average of 34.8±3.04 years;

[0032] Among them, the tuberculous ulcer group (CTB) and the control group (CG) had no significant difference in gender and age.

Embodiment 2

[0033] Example 2: Design and synthesis of PCR primer combinations for diagnosing tuberculous ulcer

[0034] According to the human CXCL9 gene (accession number: BC063122.1, gene sequence shown in SEQ ID NO.1) and human CXCL10 gene (accession number: BC010954.1, gene sequence shown in SEQ ID NO.2) published by GenBank RT-qPCR amplification primers were designed for the coding sequence and synthesized by Shanghai Sangon Bioengineering Co., Ltd. After several times of debugging and verification, the final primer sequence was determined as follows:

[0035] CXCL9 gene:

[0036] Forward primer CXCL9-F: 5'-CAGTAGTGAGAAAGGGTCG-3' (SEQ ID NO.3);

[0037] Reverse primer CXCL9-R: 5'-CATCTGCTGAATCTGGGTT-3' (SEQ ID NO.4);

[0038] CXCL10 gene:

[0039] Forward primer CXCL10-F: 5'-AGAACTGTACGCTGTACCTG-3' (SEQ ID NO.5);

[0040] Reverse primer CXCL10-R: 5'-GTAGCAATGATCTCAACACG-3' (SEQ ID NO.6);

[0041] The primer sequence of the internal reference gene U6 is:

[0042] Forward primer ...

Embodiment 3

[0044] Example 3: Correlation between the expression of CXCL9 and CXCL10 genes in the plasma of the tuberculous ulcer group and the control group and the tuberculous ulcer

[0045] 1) Plasma sample processing

[0046] Plasma samples are extracted from the whole blood samples of the tuberculous ulcer group (CTB) and the normal control group (CG) in Example 1. The specific method is: get 1ml of whole blood samples and put them into anticoagulant tubes such as heparin sodium tubes and EDTA tubes. In the tube, let it stand still or centrifuge (4°C, 2500rpm for 5min), then take the upper plasma sample and put it into a 1.5ml EP tube and store it in a 4°C refrigerator.

[0047] 2) Extraction of plasma total RNA of tuberculous ulcer group (CTB) and control group (CG)

[0048] Using TRIzol TM LS Reagent reagent (purchased from Thermo Fisher Scientific, catalog number: 10296010) was used to extract total RNA from each plasma sample extracted in step 1), and the experimental operatio...

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Abstract

The invention discloses a PCR primer combination for diagnosing tuberculous ulcer based on RT-qPCR, a kit and an application of the kit, concretely discloses applications of a CXCL9 gene and a CXCL10gene in the preparation of products for diagnosing tuberculous ulcer, further discloses PCR primers of the CXCL9 gene and the CXCL10 gene for RT-qPCR diagnosis of the tuberculous ulcer, and also discloses the PCR primer combination and an application thereof. U6 is taken as a reference gene, and the expression level of the CXCL9 gene and / or the CXCL10 gene is detected based on an RT-qPCR technology. When the 2<-deltaCt> value of the CXCL9 in blood plasma to be detected is greater than or equal to 0.112 and / or the 2<-deltaCt> value of the CXCL10 is greater than or equal to 4.014, it is shown that the patient suffers from the tuberculous ulcer or the risk of suffering from the tuberculous ulcer exists; and when the 2<delta-Ct> value of the CXCL9 in the blood plasma to be detected is less than 0.112 and the 2<-deltaCt> value of the CXCL10 is less than 4.014, the possibility of not suffering from the tuberculous ulcer is high. Compared with a traditional detection means, the kit has is more specific and sensitive in gene diagnosis, and provides a new tool for rapid and accurate diagnosis of tuberculous ulcer.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a PCR primer combination, a kit and an application thereof for diagnosing tuberculous ulcer. Background technique [0002] Tuberculous ulcer (Tuberculous lymphadenitis) refers to Mycobacterium tuberculosis (MTB), Mycobacterium bovis and Bacillus Calmette-Guerin (BCG) invading local tissues of the body through various transmission routes, causing necrosis of soft tissue, subcutaneous and skin around the lesion, and eventually The wound formed by liquefaction rupture belongs to extrapulmonary tuberculosis, accounting for about 30% to 40% of all extrapulmonary tuberculosis, and is the most common specific infectious wound in clinical practice. Early diagnosis and timely intervention are effective means of tuberculosis control. However, due to the difference in the number, virulence, transmission route, pathogenesis mode, and body immunity of mycobacteria, patien...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/158C12Q2561/113C12Q2563/107C12Q2545/114C12Q2521/107
Inventor 黄子慧顾宏伟钱佳燕张国英刘万里施金土李欣王旭朱群朱敏颜延凤陈磊垚赵璟张晓阳孙佳玥陈晓钰
Owner 南京市中西医结合医院
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