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Fluorescent polymerase chain reaction (PCR) kit for detecting chlamydia trachomatis infection by SYBR Green method

A Chlamydia trachomatis and kit technology, applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve problems such as unsuitable for clinical application, time-consuming and expensive, and complicated experimental methods, and achieve DNA Specific spectroscopic technology for hybridization, simple operation steps, and good specificity

Inactive Publication Date: 2011-06-15
上海裕隆医学检验所股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, the culture test method of Chlamydia trachomatis is a classic test method, but it is not suitable for clinical application due to the need for special equipment and reagents and the complexity of the experimental method, high technical requirements, time-consuming and high cost

Method used

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  • Fluorescent polymerase chain reaction (PCR) kit for detecting chlamydia trachomatis infection by SYBR Green method
  • Fluorescent polymerase chain reaction (PCR) kit for detecting chlamydia trachomatis infection by SYBR Green method
  • Fluorescent polymerase chain reaction (PCR) kit for detecting chlamydia trachomatis infection by SYBR Green method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: the preparation of kit

[0035] 1. Primer design and synthesis

[0036] Use Primer premier5.0 to design upstream and downstream primers for the major outer membrane protein gene of CT (studies have shown that the outer membrane major protein gene has excellent specificity). The primers were synthesized by a professional company (Shenggong), and the primers were purified by PAGE. The amplified sequence is shown in Table 1:

[0037] Table 1. Specific primer sequences

[0038] sequence name

Oligonucleotide sequence (5'-3')

Base length (bp)

CT forward primer

CCATGAGTGGCAAGCAAGTTTAGC

24

CT reverse primer

CAGCGATGGTCGGGTTTAGAG

21

[0039] 2. Preparation of CT plasmid positive reference substance

[0040] In this example, the CT plasmid was used as a positive template for the positive control.

[0041] According to the primers for amplifying the CT-specific sequences synthesized in step 1, select a sequ...

Embodiment 2

[0046] Embodiment 2: the use of kit

[0047] 1. Sample extraction

[0048] Use the DNA extraction solution to extract the CT virus nucleic acid in the sample to be tested

[0049] 1) Sample pretreatment: wipe off excess secretions from the cervix, use a cotton swab soaked in normal saline to cling to the cervical mucous membrane and rotate it for 2 weeks to obtain secretions and exfoliated cells, and put the cotton swab after sampling Rinse fully in an EP tube with 1ml of sterile saline, and squeeze dry by sticking to the wall. The CT virus was extracted by alkaline lysis.

[0050] Lysis solution formula: 60mmol / L TrisHCl, pH 8.0; 0.5% SDS; 200mmol / L NaCl;

[0051] 2) Operation steps: Take 500 μl of secretions, mix and centrifuge at 13,000 rpm for 10 minutes, discard the supernatant; add 50 μl of lysate, incubate at 100°C for 20 minutes, centrifuge at 13,000 rpm for 1 minute, and take 1 μl of the supernatant for PCR reaction.

[0052] 2. Preparation of CT positive control ...

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Abstract

The invention discloses a fluorescent polymerase chain reaction (PCR) kit for detecting chlamydia trachomatis infection by an SYBR Green method and belongs to the field of in-vitro nucleic acid diagnosis kits. The kit comprises a negative reference and control substance, a negative reference substance, fluorescent PCR reaction liquid, Taq enzyme and deoxyribonucleic acid (DNA) extraction liquid. The kit comprises a PCR reaction system in which a fluorescent PCR technology is taken as basis, forward and reverse primers and fluorescent dye SYBR Green aiming at the specific sequence of chlamydia trachomatis, can detect chlamydia trachomatis infection in a clinical sample conveniently and quickly, and has high specificity and great clinical value for early detection of the chlamydia trachomatis.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, and relates to a fluorescent PCR (polymerase chain reaction) kit for detecting chlamydia trachomatis infection in clinical samples. Background technique [0002] Chlamydia trachomatis (Chlamydia trachomatis, CT) is a Gram-negative pathogen without the ability to synthesize the high-energy compound ATP, which must be provided by the host cell, so it is an energy parasite. Chlamydia is a prokaryotic microorganism that can pass through the cell filter, has a unique developmental cycle, and is strictly intracellular. It is larger than viruses and smaller than bacteria. It is spherical in shape with a diameter of only 0.3 to 0.5 mm. CT is one of the pathogens of important sexually transmitted diseases. It not only causes vaginal and urinary tract infections; ascending reproductive tract infections may also involve the endometrium, fallopian tubes, and adjacent pelvic structures, leading ...

Claims

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Application Information

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IPC IPC(8): C12Q1/04G01N21/64C12Q1/68
Inventor 穆海东汪宁梅穆宇豪黎飒
Owner 上海裕隆医学检验所股份有限公司
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