Primer, probe and kit for detecting pneumocystis carinii and toxoplasma gondii and detection method thereof

A kit and Toxoplasma gondii technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of missed diagnosis and low sensitivity of microscopic examination

Active Publication Date: 2020-11-13
JIANGSU BIOPERFECTUS TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection rate of microscopic examination of alveolar lavage fluid has been greatly improved compared with sputum specimens, but the sensitivity of microscopic examination is still low, which may easily lead to missed diagnosis

Method used

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  • Primer, probe and kit for detecting pneumocystis carinii and toxoplasma gondii and detection method thereof
  • Primer, probe and kit for detecting pneumocystis carinii and toxoplasma gondii and detection method thereof
  • Primer, probe and kit for detecting pneumocystis carinii and toxoplasma gondii and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Extraction of total DNA from human sputum or alveolar lavage fluid samples:

[0077] 1. Pretreatment of sputum or alveolar lavage fluid:

[0078](1) Add NaOH solution (1M) into sputum or alveolar lavage fluid (sputum: NaOH solution = 1:1, lavage fluid: NaOH solution = 2:1), and observe after 1 hour in an incubator at 60°C Whether the sputum or alveolar lavage fluid is liquefied, if it is not completely liquefied, the time can be appropriately extended until the liquefaction is complete, if it has been liquefied, the following steps can be continued;

[0079] (2) Centrifuge the liquefied sputum or alveolar lavage fluid at 8000rpm / min for 5 minutes, and remove the supernatant after centrifugation;

[0080] (3) Add 1ml of normal saline, pipette to mix, centrifuge at 8000rpm / min for 5 minutes, and remove the supernatant;

[0081] (4) Repeat step (3).

[0082] 2. Extraction of total DNA in human sputum or alveolar lavage fluid:

[0083] (1) Add 700 μl of 0.1%-...

Embodiment 2

[0092] Example 2: Primer and probe design

[0093] The DNA sequences of the mitochondrial ribosomal small subunit genes of different Pneumocystis carinii were compared to determine the conserved region, and primers and probes were designed according to the conserved region. The sequences of the primers and probes are as follows, and the length of the amplified fragment is 124bp:

[0094] Primer PJ-F: 5'-TTATGAAGTGGGCTACAGAC-3' (SEQ ID No: 1);

[0095] Primer PJ-R: 5'- CTTCAAAGAGCCGAGTTCC -3' (SEQ ID No: 2);

[0096] Probe PJ-Probe: 5'-FAM-TTTGAACGGGATTTCTGCACCCAT-TAMRA-3' (SEQ ID No: 3).

[0097] The 529bp repeat sequence of different Toxoplasma gondii was compared to determine the conserved region, and primers and probes were designed according to the conserved region. The sequences of the primers and probes are as follows, and the length of the amplified fragment of Toxoplasma gondii is 233bp:

[0098] Primer TG-F: 5'- GACTACAGACGCGATGCC -3' (SEQ ID No: 4);

[0099] Prime...

Embodiment 3

[0101] Embodiment 3: the preparation of plasmid standard

[0102] 1. Preparation and purification of target fragments:

[0103] (1) Establish a PCR amplification reaction system: 2×GoTaq Green Master Mix 10μl, primer-F 200nM, primer-R 200nM, DNA sample (the nucleotide sequences of which are shown in SEQ ID No: 13 and 14 respectively) 20-50ng , supplemented to 20 μl with sterile double distilled water.

[0104] (2) PCR amplification reaction conditions: 95°C, 5min; 95°C for 15s, 60°C for 15s, 72°C for 20s, a total of 35 cycles; 72°C for 5min, 4°C ∞.

[0105] (3) Amplified product recovery: 2% agarose gel electrophoresis was used to detect the amplified product, and the general DNA purification and recovery kit of Tiangen Bio was used to cut the gel and recover the target fragment.

[0106] 2. Digestion and purification of plasmid vectors:

[0107] (1) EcoRI and HindⅢ digestion plasmid vector pUC19 linearization enzyme digestion reaction system: HindⅢ 1μl, EcoRI 1μl, 10×M buf...

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Abstract

The invention discloses a primer, a probe, a kit and a detection method for detecting pneumocystis carinii and toxoplasma gondii, and particularly relates to a kit and a detection method for double fluorescent quantitative PCR detection of pneumocystis carinii and toxoplasma gondii. According to the invention, the primer pair and the probe are designed by utilizing conserved regions of pneumocystis carinii and toxoplasma gondii, the primer pair and the probe which can specifically amplify pneumocystis carinii and toxoplasma gondii are screened out, the sensitivity is high, the two pairs of primers and the two probes do not interfere with each other, and a sample only containing 1 copy can be accurately detected; by utilizing the primer pair, the probe and the detection method, the contentsof the pneumocystis carinii and the toxoplasma gondii can be absolutely quantified; the kit is simple in structure, safe in detection, rapid and convenient, can be used for detecting the pneumocystiscarinii and the toxoplasma gondii and tracking and monitoring the treatment effect of medicines of a patient, and is beneficial to carrying out epidemiological investigation and preventing and controlling propagation of the pneumocystis carinii and the toxoplasma gondii.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer, a probe, a kit and a detection method for the detection of Pneumocystis carinii and Toxoplasma gondii, especially for dual fluorescence quantification of Pneumocystis carinii and Toxoplasma gondii Kits and detection methods for PCR detection. Background technique [0002] Pneumocystis carinii pneumonia ( Pneumocystis pneumonia ) pathogen Pneumocystis carinii ( pneumocystis carinii , PC), which are opportunistic pathogens to humans, rarely cause disease in normal humans, and mostly occur in immunocompromised populations such as AIDS patients and transplant patients. Lung stagnation is considered to be a hallmark disease of AIDS, and more than 85% of HIV-infected persons will be affected by it in their lifetime, and it is the main cause of death from AIDS. After organ transplantation, patients take immunosuppressive agents for a long time and are prone to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6893C12Q1/6895C12Q1/6851C12Q1/06
CPCC12Q1/6851C12Q1/6893C12Q1/6895C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2563/107C12Q2545/114
Inventor 杨国威吴云栗绍刚
Owner JIANGSU BIOPERFECTUS TECH CO LTD
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