Multiplex PCR detection method for simultaneously detecting various kinds of bacteria and mycoplasmas and application thereof

A detection method and mycoplasma technology, applied in the field of genetic engineering, can solve the problems of difficulty in determining accuracy, low sensitivity and high cost, and achieve the effects of high sensitivity, fast detection and simple operation

Inactive Publication Date: 2019-12-24
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

Although next-generation sequencing tests have shown great strength over traditional methods in diagnosing rare pathogens, their sensitivity is not stronger than traditional methods for common pathogens such as Mycobacterium tuberculosis and Cryptococcus
For these common pathogens, they are often not detected by tra

Method used

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  • Multiplex PCR detection method for simultaneously detecting various kinds of bacteria and mycoplasmas and application thereof
  • Multiplex PCR detection method for simultaneously detecting various kinds of bacteria and mycoplasmas and application thereof
  • Multiplex PCR detection method for simultaneously detecting various kinds of bacteria and mycoplasmas and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] (1) Extraction of bacterial and mycoplasma genomic DNA

[0079] To collect the infected samples from the subjects to be tested, commercially available bacterial and mycoplasma genomic DNA extraction kits were used, and the specific operation process was carried out according to the instructions.

[0080] (2) Primer design

[0081] Acinetobacter baumannii, Proteus mirabilis, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas maltophilia, Pseudomonas aeruginosa, Pseudomonas cepacia, Escherichia coli, Corresponding sequences from Neisseria meningitidis, Klebsiella pneumoniae, Staphylococcus hominis, Legionella pneumophila, Staphylococcus aureus, Haemophilus influenzae, MRSA, Mycobacterium tuberculosis and Mycoplasma pneumoniae , design amplification primer probes (Table 1), to ensure that each pair of primers and corresponding probes can amplify the corresponding pathogenic microorganisms, and do not amplify non-specifically with othe...

Embodiment 2

[0093] Specificity experiment:

[0094] Bacteria Genomic DNA Extraction Kit was used to extract Acinetobacter baumannii, Proteus mirabilis, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas maltophilia, Pseudomonas aeruginosa, Pseudomonas cepacia, Escherichia coli Escherichia, Neisseria meningitidis, Klebsiella pneumoniae, Staphylococcus hominis, Legionella pneumophila, Staphylococcus aureus, Haemophilus influenzae, MRSA, Mycobacterium tuberculosis, and pneumonia For mycoplasma DNA, the operation steps were carried out according to the instructions provided with the DNA extraction kit. The detection channels are divided into five groups, and the grouping conditions are the same as those in Table 2. Taking the first group as an example, add corresponding primer probes to the wells in the same row of the 96-well plate (only the 4 primer probes of the first group are added to each tube). , in this way, rows 1-5 correspond to the primer prob...

Embodiment 3

[0101] Sensitivity experiment

[0102] 18 kinds of bacteria and mycoplasma (pathogenic microorganism) DNA prepared in embodiment 2 are carried out tenfold serial dilution, 10 2 -10 5 copies / ul, using the kit provided by the invention to amplify the above-mentioned diluted samples respectively, the detection sensitivity results of 18 kinds of pathogenic microorganisms Figure 1-Figure 18 As shown, among them, Figure 1-Figure 18 The R of the standard curve of the detection results of 18 kinds of pathogenic microorganisms 2 The values ​​are 0.9926 (Acinetobacter baumannii), 0.9961 (Enterococcus faecalis), 0.9988 (Escherichia coli), 0.9962 (Klebsiella pneumoniae), 0.9862 (Proteus mirabilis), 0.9999 (Pseudomonas aeruginosa ), 0.9983 (Pseudomonas cepacia), 0.9938 (Pseudomonas maltophilia), 0.9867 (Streptococcus pneumoniae), 0.9921 (Staphylococcus epidermidis), 0.9874 (Neisseria meningitidis), 0.9876 (Staphylococcus hominis) , 0.9963 (Legionella pneumophila), 0.9900 (Staphylococ...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to a primer and probe combination for simultaneously detecting various kinds of bacteria and mycoplasmas. The invention also provides a multiplex PCR detection method for simultaneously detecting various kinds of bacteria and mycoplasmas. When the method is used, various kinds of bacteria and mycoplasmas can be detected at the same time; multiplex detection is realized; the sensitivity is high; high speed and convenience are realized; and the batch detection on samples can be realized. The invention also provides a kit for simultaneously detecting various kinds of bacteria and mycoplasmas and application thereof. Various technical problems of common infectious pathogenic microorganism detection in clinics are solved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a multiple PCR detection method for simultaneously detecting multiple bacteria and mycoplasma and its application. Background technique [0002] At present, the commonly used methods for clinical detection of bacteria include bacterial culture, PCR method, first-generation sequencing and next-generation sequencing. Bacterial culture is the gold standard for clinical detection of bacteria, but this method requires drug susceptibility testing, which usually takes 1-3 days to obtain the final test results, and batch samples cannot be processed at the same time. Therefore, the detection cost of this method is high, the cycle is long, the operation is cumbersome, and it is difficult to meet the clinical needs. The PCR method can geometrically increase the copy number of a given nucleic acid sequence in a short period of time. It has high sensitivity and specificity, but i...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12Q1/686C12R1/35C12R1/45C12R1/46C12R1/38C12R1/385C12R1/19C12R1/22C12R1/44C12R1/01C12R1/445C12R1/21C12R1/32
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
Inventor 陈江坡胖铁良卢孟孟马蒙蒙孙晋华李建中
Owner 廊坊诺道中科医学检验实验室有限公司
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