Nucleic acid reagent, kit and system for detecting lower respiratory tract infection bacteria

A respiratory and kit technology, applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc., can solve problems such as poor ability to produce antibodies, inability to meet fast detection, and inapplicability of early diagnosis, etc. to save time

Active Publication Date: 2020-03-20
CHINA JAPAN FRIENDSHIP HOSPITAL +1
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AI Technical Summary

Problems solved by technology

[0003] The isolation and culture method is a common method for detecting bacteria in lower respiratory tract infections, and its test results are often used as the "gold standard" for the diagnosis of respiratory tract bacterial infections. However, the specificity and sensitivity of this method are poor, and it is not suitable for mild infections. Early diagnosis, unable to meet the needs of rapid detection
[0004] Serological testing is also a common method for detecting bacteria in lower respiratory tract infections. This method uses the relevant antigens produced by the human body as the detection target. However, the production of antigens is greatly affected by the course of the disease. It can only be detected after a week, which cannot meet the needs of rapid detection; moreover, for infants and young children, because the immune function is not yet fully developed, the ability to produce antibodies is poor, and there is a great possibility of false negatives in serological testing. The result is not accurate enough

Method used

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  • Nucleic acid reagent, kit and system for detecting lower respiratory tract infection bacteria
  • Nucleic acid reagent, kit and system for detecting lower respiratory tract infection bacteria
  • Nucleic acid reagent, kit and system for detecting lower respiratory tract infection bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1 detection method and detection result judgment

[0073] 1. Primer and probe synthesis

[0074] Sequence synthesis was performed according to the primer sequences shown in Table 1 and the probe sequences shown in Table 2. In the probe, FAM is 6-carboxyfluorescein, CY5 is 5H-indocyanine, ROX is 6-carboxy-X-rhodamine, VIC is a dye purchased from ABI, and BHQ1 and BHQ2 are quenching groups.

[0075] Table 1

[0076]

[0077] Table 2

[0078]

[0079]

[0080] 2. Sample processing

[0081] The liquefied sputum sample was centrifuged at 13,000 rpm for 5 minutes, and the centrifuged precipitate was mixed with 200 μL of normal saline, then centrifuged and washed at 13,000 rpm for 5 minutes, and repeated twice. After centrifugation and washing, remove the supernatant, add 50 μL nucleic acid extraction solution and 10 μL internal standard control to the obtained precipitate, shake and mix, heat in a metal bath or boiling water bath at 100 °C for 10 minut...

Embodiment 2

[0099] Embodiment 2 minimum detection limit verification

[0100] Test samples for evaluation: Use commercial kits to extract Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter cloacae, Genomic DNA from Burkholderia cepacia, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, and Enterococcus faecalis, and quantified at 1 × 10 6 copies / mL, and then serially diluted to obtain a concentration of 1×10 5 copies / mL, 1×10 4 copies / mL, 1×10 3 copies / mL, 1×10 2 Copies / mL evaluation template.

[0101] According to the detection method of Example 1, the evaluation templates of each concentration were detected respectively, and the detection was repeated 20 times for each concentration gradient, and the average value was taken as the final detection result, as shown in Table 3.

[0102] table 3

[0103]

[0104] Note: "+" means positive, n / 20 means the detection ...

Embodiment 3

[0106] Example 3 specificity verification

[0107]Select human coronavirus (purchased from Wuhan Institute of Virology, Chinese Academy of Sciences, number 2233), cytomegalovirus (purchased from Wuhan Institute of Virology, Chinese Academy of Sciences, number 303), enterovirus (purchased from Wuhan Institute of Virology, Chinese Academy of Sciences, number 977) ), measles virus (purchased from Wuhan Institute of Virology, Chinese Academy of Sciences, number 1513), human metapneumovirus (from the General Hospital of the Chinese People's Liberation Army, number 1768), rhinovirus (purchased from Wuhan Institute of Virology, Chinese Academy of Sciences, number 2343 ), avirulent Mycobacterium tuberculosis (purchased from China Medical Culture Collection Center, No. 93009), Neisseria meningitidis (from the General Hospital of the Chinese People's Liberation Army, No. 21123), Streptococcus pyogenes (from PLA General Hospital, No. 25341), Streptococcus salivarius (from the Chinese Peo...

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Abstract

The disclosure relates to a nucleic acid reagent for detecting lower respiratory tract infection bacteria. The nucleic acid reagent comprises primers shown in SEQ ID NO.1-24 and probes shown in SEQ IDNO.27-38 which are stored in a respective and independent manner or in a mutual and arbitrary mixing manner. Through the primers and probes described above, the nucleic acid reagent, a kit, a systemand a method for detecting 12 types of lower respiratory tract infection bacteria such as streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, pseudomonas aeruginosa, klebsiella pneumonia, acinetobacter baumannii, enterobacter cloacae, burkholderia cepacia, escherichia coli, staphylococcus aureus, enterococcus faecium, and enterococcus faecalis are established, at the same time, internal reference genes are added as controls, the above 12 types of bacteria can be quantitatively detected through a double standard curve method, the uniform quantification treatment of samplescan be performed, the fast, comprehensive, sensitive, specific and automatic detection result determination is realized, and sensitivity, specificity, and simplicity of simultaneously detecting the above detection target genomes are significantly improved.

Description

technical field [0001] The present disclosure relates to the field of biotechnology, in particular to a nucleic acid reagent, kit and system for detecting lower respiratory tract infection bacteria. Background technique [0002] Community Acquired Pneumonia (CAP) refers to infectious lung parenchyma (including alveolar wall, that is, lung interstitium in a broad sense) inflammation caused by bacteria, viruses, chlamydia, mycoplasma and other microorganisms outside the hospital, including Pneumonia that develops during the incubation period after admission to the hospital due to pathogenic infection with a definite incubation period. The composition and drug resistance of CAP pathogens are significantly different in different countries and regions, and have changed over time. Hospital acquired pneumonia (HAP), also known as nosocomical pneumonia (NP), refers to a patient who does not exist at the time of admission and is not in the incubation period of infection, but occurs ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12N15/11C12Q1/04C12Q1/10C12Q1/14C12R1/445C12R1/46C12R1/21C12R1/01C12R1/385C12R1/22C12R1/19
CPCC12Q1/6851C12Q1/689C12Q2600/16C12Q2600/166C12Q2531/113C12Q2545/101C12Q2563/107C12Q2537/143
Inventor 王晓艳曹彬傅成波刘颖梅王雷鲁炳怀黎斌斌徐丹丹王月张志强
Owner CHINA JAPAN FRIENDSHIP HOSPITAL
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