Kit for simultaneously detecting streptococcus pneumoniae, legionella pneumophila and Moraxella catarrhalis

A technology for Streptococcus pneumoniae and Legionella pneumophila, applied in the field of fluorescent PCR detection, can solve the problems of long time, easy missed diagnosis, high nutritional requirements, and achieve the effect of ensuring timeliness, fast detection method, and good specificity

Pending Publication Date: 2021-03-12
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional method of isolation without culture needs to be cultured and isolated to identify the strain, which takes a long time, and the growth conditions of Legionella pneumophila are harsh, the nutritional requirements are high, time-consuming and labor-intensive, it is not conducive to rapid detection, and it has certain requirements for operators. risk, thus limiting its clinical application
The immunoassay detection reagent is aimed at the antigen in the sample or the specific antibody produced by the body caused by the pathogen, and there is a window period for antibody detection, so it is easy to miss the diagnosis in the early stage of the disease
In addition, although the serological method is mature and commercial kits are available, the sensitivity and specificity need to be improved. At the same time, it is necessary to detect double serum in the acute phase and the convalescent phase to be diagnostically meaningful and it is easy to miss the diagnosis.
However, the fluorescent PCR detection in the prior art is difficult to realize multiple detection of SP, LP, and MC due to reasons such as primer specificity.

Method used

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  • Kit for simultaneously detecting streptococcus pneumoniae, legionella pneumophila and Moraxella catarrhalis
  • Kit for simultaneously detecting streptococcus pneumoniae, legionella pneumophila and Moraxella catarrhalis
  • Kit for simultaneously detecting streptococcus pneumoniae, legionella pneumophila and Moraxella catarrhalis

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preparation example Construction

[0049] 3. Preparation of quality control products

[0050] Positive quality control: Mix cultures of Streptococcus pneumoniae, Legionella pneumophila and Moraxella catarrhalis 1:1:1; dilute 1000 times, aliquot 0.65mL. Negative quality control: β-globin pseudovirus diluted and mixed with virus preservation solution, aliquoted into 0.65mL.

Embodiment 1

[0052] Embodiment 1 simultaneously detects the preparation of the kit of Streptococcus pneumoniae, Legionella pneumophila and Moraxella catarrhalis

[0053] The kit includes PCR reaction solution, negative quality control, and positive quality control;

[0054] Wherein, the components of the PCR reaction solution are shown in Table 2:

[0055] Table 2 The components and concentrations in the PCR reaction solution

[0056] components volume in each reaction system reaction buffer 10 μL dNTPs (10mM) 2μL MLV enzyme (200U / μL) 0.1μL Taq enzyme (5U / μL) 0.3μL MgCl 2 (50mM)

2μL SEQ ID NO: 1 1μL SEQ ID NO: 2 1μL SEQ ID NO: 3 0.5μL SEQ ID NO: 4 1μL SEQ ID NO: 5 1μL SEQ ID NO: 6 0.5μL SEQ ID NO: 7 1μL SEQ ID NO: 8 1μL SEQ ID NO: 9 0.5μL SEQ ID NO: 11 0.5μL SEQ ID NO: 12 0.5μL SEQ ID NO: 13 0.25 μL Sterilized purified water Up To 25μL

[0057] P...

Embodiment 2

[0059] The detection method of embodiment 2 kit of the present invention

[0060] Mix the human sputum sample with a pipette, take out 200 μL of sputum, add 4 times the volume of 4% NaOH (self-prepared), shake well, and leave it at room temperature for 30 minutes to liquefy (if the sputum is still very viscous, you can increase the storage time Until the sputum is liquefied), take 0.5mL sample to a 1.5mL centrifuge tube, then add 0.5mL 4% NaOH and leave it at room temperature for 10 minutes, then centrifuge at 13000rpm for 5 minutes. Add 1 mL of sterile saline to the precipitate, mix well, and centrifuge at 13,000 rpm for 5 minutes; repeat the washing once more, and remove the supernatant. Add 200 μL of bacterial lysate (from Beijing Biolab Technology Co., Ltd.) to the precipitate, mix well, bathe in water at 100°C for 10 minutes, and centrifuge at 13,000 rpm for 5 minutes for later use.

[0061] Streptococcus pneumoniae, Legionella pneumophila and Moraxella catarrhalis nucle...

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Abstract

The invention relates to the technical field of fluorescent PCR detection, in particular to a kit for simultaneously detecting streptococcus pneumoniae, legionella pneumophila and Moraxella catarrhalis. A fluorescent PCR detection system comprises primers of specific conserved sequences of streptococcus pneumoniae, legionella pneumophila and Moraxella catarrhalis and a TaqMan fluorescent probe, and the DNA sequence of the fluorescent PCR detection system is SEQ ID NO: 1-9. The kit has the advantages that multiple fluorescent quantitative PCR detection technologies are utilized, the purpose ofsimultaneously detecting three respiratory tract pathogens including streptococcus pneumoniae, legionella pneumophila and Moraxella catarrhalis is is achieved, the detection method is rapid and accurate, timeliness, specificity and sensitivity of detection are guaranteed, and the kit has important application value in the fields of disease monitoring, clinical diagnosis and the like.

Description

technical field [0001] The invention relates to the technical field of fluorescent PCR detection, in particular to a kit for simultaneously detecting Streptococcus pneumoniae, Legionella pneumophila and Moraxella catarrhalis. Background technique [0002] Respiratory tract infection is one of the most common infectious diseases worldwide and an important issue affecting human health. Respiratory tract infection includes upper respiratory tract infection and lower respiratory tract infection. There are many pathogens that can cause respiratory tract infection, including bacteria, viruses, mycoplasma, chlamydia, etc. The symptoms caused by these pathogens are similar, and it is difficult to distinguish them based on clinical symptoms. Lower respiratory tract infection refers to the inflammation of the terminal airways, alveoli and pulmonary interstitium, which can be caused by disease microorganisms, physical and chemical factors, immune injury, allergies and drugs. Among the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12N15/11C12Q1/14C12Q1/04C12R1/01C12R1/46
CPCC12Q1/689C12Q1/6851C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2545/101C12Q2561/101C12Q2563/107
Inventor 吕月庆高利飞高歌杜美王玮李振红付光宇
Owner AUTOBIO DIAGNOSTICS CO LTD
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