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Composition, kit, use and method for detecting deep infection fungi

A composition and kit technology, applied in the field of molecular biology detection, can solve the problems of inability to guarantee sensitivity, poor specificity, low sensitivity, etc., and achieve the effects of avoiding false positives and environmental pollution, good specificity and high sensitivity

Active Publication Date: 2020-07-28
SANSURE (SHANGHAI) GENE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, although some studies try to use fluorescent quantitative PCR method to detect Pneumocystis, Cryptococcus neoformans, and Candida albicans to solve the consumption of conventional detection methods (such as staining microscopy, pathological examination, blood culture and agglutination tests, etc.) The problem of time length and low sensitivity, for example, CN110551840A discloses a nucleic acid reagent for detecting invasive fungi, the nucleic acid reagent used in the method corresponds to primers and probes of three kinds of fungi, and the three kinds of fungi are detected
However, these methods still have defects such as low sensitivity (detection rate is only 40-60%), poor specificity (non-specific amplification of Enterococcus faecalis and botulinum), especially in one-step detection methods , and the sensitivity cannot be guaranteed

Method used

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  • Composition, kit, use and method for detecting deep infection fungi
  • Composition, kit, use and method for detecting deep infection fungi
  • Composition, kit, use and method for detecting deep infection fungi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1, primers and probes used in the present invention

[0071] The primers and probes used in the present invention are as follows:

[0072] Cryptococcus neoformans upstream primer (SEQ ID NO: 1): 5'- TTGGACTTGGATTTGGGTGTTT-3';

[0073] Cryptococcus neoformans downstream primer (SEQ ID NO:2): 5'-CTTATTACGCCGGGCTGACA-3';

[0074] Cryptococcus neoformans probe (SEQ ID NO:3): 5'-CCTGCAAAGGACGTCGGCTCGC-3';

[0075] Human Pneumocystis upstream primer (SEQ ID NO:4): 5'-AGCCGAGTTCCAGGCACTTA-3';

[0076] Human Pneumocystis downstream primer (SEQ ID NO:5): 5'-GCTACAGACGTGCTGCAAAATT-3';

[0077] Human Pneumocystis probe (SEQ ID NO:6): 5'-CTCCGACTTCCATCATTGCATCCCA-3';

[0078] Candida albicans upstream primer (SEQ ID NO:7): 5'-ACTTCAGGTACCGTTGATTTCCA-3';

[0079] Downstream primer for Candida albicans (SEQ ID NO:8): 5'-TTTCACGAACACGAATTTCACAT-3';

[0080] Candida albicans probe (SEQ ID NO:9): 5'-TCTCCGTTCCTGCTTCGGAATTCCTTTA-3';

[0081] Internal standard upstream p...

Embodiment 2

[0085] Embodiment 2, the method for detecting deep infection fungus and typing

[0086] The detection samples of the present invention are throat swabs, sputum, alveolar lavage fluid, blood, etc., and the following operations are performed in the sample processing room:

[0087] (1) After the sample is centrifuged, remove the supernatant, add a nucleic acid release agent to the pellet, mix thoroughly, and let stand for 5-10 minutes (no need to extract or purify nucleic acid);

[0088] (2) Prepare 50 µl of fluorescent PCR amplification reaction system according to the following components;

[0089] (3) On the PCR amplification instrument, the primers and probes specifically amplify the target sequence of the gene to be tested;

[0090] (4) Result analysis.

[0091] The real-time fluorescent PCR reaction system is configured as follows:

[0092]

[0093] The PCR amplification program is set as follows:

[0094] .

[0095] Result analysis:

[0096] 1) The target detect...

Embodiment 3

[0104] Embodiment 3, the detection result of composition test clinical sample of the present invention

[0105] Use the composition described in Example 1 of the present invention (including all primers and probes) to be configured into a PCR tube to form a system. According to the method described in Example 2, 80 cases of clinical samples were tested, and the final test 12 cases of Candida albicans, 8 cases of Cryptococcus neoformans, and 6 cases of human Pneumocystis were positive. Use clinical samples to perform multiple PCR detection on the Hongshi fluorescence quantitative PCR instrument, and the results of positive samples are as follows: Figure 1~4 As shown, the results of all channels of the negative sample are as follows Figure 5 shown. Good amplification curves can be detected in each channel, indicating that the composition of the present invention can detect and type the fungi that cause deep infections without false positives.

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Abstract

The invention relates to the field of molecular biology detection, and more specifically to the detection of Candida albicans, Cryptococcus neoformans, and Pneumocystis jiroveci. The invention provides a composition for detecting the above-mentioned fungi; and at the same time also provides a kit containing the composition, a use of the composition, and a method for detecting and typing deep infection fungi. The composition of the invention can simultaneously detect the three fungi in one tube and perform typing by combining with a fluorescent probe method, and has the advantage that the costis low, the throughput is high, time-consuming is short, the operation is simple and convenient, and false positives caused by crossover between samples and environmental pollution are avoided. Further, the composition of the invention is adapted to a one-step method, the sensitivity and specificity are ensured while the detection time is saved and the detection efficiency is improved.

Description

technical field [0001] The invention belongs to the field of molecular biology detection; specifically, it relates to the detection of deep infection fungi, more specifically, it relates to the detection of Candida albicans, Cryptococcus neoformans, and Pneumocystis of human origin. Background technique [0002] The morbidity and mortality of deep mycoses in tumor patients receiving chemotherapy, organ transplantation patients and immunosuppressed patients infected with AIDS are increasing year by year. The main fungi that infect deep mycosis include the following: [0003] Pneumocystis carinii (Pneumocystis carinii) pneumocystis carinii ), those who parasitize the human body are called Pneumocystis jirovecii ( pneumocystis jiroveci ), also known as human Pneumocystis. Pneumocystis pneumonia (PCP) is a severe and fatal pneumonia caused by Pneumocystis parasites in the lungs. PCP is prone to occur in immunocompromised populations such as AIDS, leukemia, organ transplanta...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6893C12Q1/6851C12Q1/06C12N15/11C12R1/645C12R1/725
CPCC12Q1/6851C12Q1/6893C12Q1/6895C12Q2600/166C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 孙青芝刘佩珊任小梅谭德勇刘佳邓中平戴立忠
Owner SANSURE (SHANGHAI) GENE TECH LTD
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