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A primer, probe and kit for quantitative pcr to detect all 11 species of chlamydia

A chlamydia and kit technology, applied in the direction of microorganisms, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of low specificity and sensitivity, limited sensitivity, inability to distinguish immunization and natural infection, etc., and achieve high specificity The effect of high quality, high sensitivity and convenient operation

Inactive Publication Date: 2016-02-24
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional methods mainly rely on isolation and culture. Commonly used methods include cell culture, chicken embryo isolation and mouse inoculation. However, this method takes a long time, has limited sensitivity, and the detection of chlamydia species is limited, resulting in missed detection; ELISA method is accurate and fast Easy to use, suitable for batch sample detection, but cross-reaction with other microorganisms, can not distinguish a variety of chlamydia; IHA and CF are characterized by convenience, low equipment requirements, but low specificity and sensitivity, unable to distinguish immunization and natural infection, the antibodies of recovered animals may also exist and easily cause false positives; the single PCR method reported so far cannot detect multiple chlamydia at the same time; the existing multiplex PCR method can simultaneously amplify more than one sequence to detect different species of chlamydia purpose of the genus, but lacks standardized reagents

Method used

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  • A primer, probe and kit for quantitative pcr to detect all 11 species of chlamydia
  • A primer, probe and kit for quantitative pcr to detect all 11 species of chlamydia
  • A primer, probe and kit for quantitative pcr to detect all 11 species of chlamydia

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preparation example Construction

[0106] The DNA template preparation process is equipped with negative and positive controls for DNA extraction, and the subsequent PCR amplification objects include the DNA template of the sample to be tested, negative and positive controls, and quantitative standard reagents (each 10 μl standard contains 10 4 , 10 3 , 10 2 , 10 1 , 1 copy of 23SrRNA gene).

[0107] 7. Agarose Gel Electrophoresis

[0108] Prepare 2% agarose gel, take 5 μl PCR amplification product, and use SafeDNA Gel Stain staining. Observed under ultraviolet light, all 11 species of Chlamydia had target bands at 170 bp, and the species of Chlamydia with similar or overlapping melting curves could be typed by PCR product sequencing.

Embodiment 1

[0109] Embodiment 1: PCR method amplifies the nucleic acid of Chlamydia pneumoniae ATCC strain

[0110] Chlamydia pneumoniae standard strain (ATCC strain) was harvested by conventional Hep-2 cell culture, and 200 μl of sample was taken for nucleic acid purification and PCR amplification described in the present invention. Through the quantitative conversion of the synthetic standard, it is determined that each ml harvested sample contains about 5×10 8 Copy the 23SrRNA gene.

Embodiment 2

[0111] Embodiment 2: Detection of poultry throat and cloacal swab samples by PCR method

[0112] A total of 4045 poultry throat and cloacal swab samples were collected from 24 provinces and cities across the country. Immediately after the swab sample was collected, it was stored in a 1.5ml EP tube containing 500μl nucleic acid protectant, and then the nucleic acid was purified by the method of the present invention, and finally eluted in 200μl ElutionBuffer eluent, which was used as the amplification template of PCR. Chlamydia nucleic acid standard 10 6 / 10μl of sample with ddH 2 O diluted to 10 4 / 10μl and 10 3 / 10 μ l as a positive control, detect the nucleic acid of the swab sample with specific primers and probes of the present invention, classify according to the melting curves of different species of Chlamydia, T m Several similar or overlapping Chlamydia were typed by PCR product sequencing method. Using the present invention, we detected 5 different kinds of Chlam...

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Abstract

The invention provides primers, probes and a kit for quantitative PCR (polymerase chain reaction) for detecting 11 types of chlamydiae. The sequences of the primers are shown by SEQ ID No.1, 2 and 3, the probes 1 are shown by SEQ ID No.4, 5 and 6, and the primers and the probes together with a PCR reaction solution form a quantitative PCR detection kit. The primers (including 2 sense primers and 1 reverse primer aiming at the 11 types of chlamydiae) and the probes (3 probes) are designed according to a conservative region of 23S rRNA genes of the chlamydiae. The primers and the probes of PCR are ingeniously designed, so that the nucleic acids of all 11 types of the chlamydiae can be specially amplified, and typing can be performed according to a Tm value of a melting curve, so that the primers, the probes and the kit are suitable for detecting a lot of clinical samples.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a primer, a probe and a kit for quantitative PCR, which are based on the 23SrRNA gene sequence of chlamydia and are suitable for real-time FRET-PCR detection of all 11 kinds of chlamydia. Background technique [0002] Chlamydia is Gram-negative, a pathogen between bacteria and viruses, belongs to strict intracellular parasitism, and exists widely in nature. Chlamydia can infect mammals, birds and humans, and can cause a variety of animal and human diseases, and different species of Chlamydia can also spread between different hosts, which is very important for the healthy development of the breeding industry, the import and export of animal products, and human health. Both public health and safety fields have caused huge economic losses. Among them, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia abortus and Chlamydia psittaci are important zoonotic pathogens: Chlam...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 王成明李静B·卡腾巴克郭伟娜龚建森
Owner YANGZHOU UNIV