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Kit, reaction system and method for detecting pneumocystis jirovecii and drug resistance of pneumocystis jirovecii

A reaction system and technology for spore bacteria, which are applied in the directions of microorganism-based methods, biochemical equipment and methods, and the determination/inspection of microorganisms, which can solve the problems of lack of established drug resistance detection methods and low attention

Inactive Publication Date: 2020-12-11
上海捷诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And at present, the clinical attention to drug-resistant strains of Pneumocystis is low, and there is still no simultaneous standardized and effective drug resistance detection method

Method used

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  • Kit, reaction system and method for detecting pneumocystis jirovecii and drug resistance of pneumocystis jirovecii
  • Kit, reaction system and method for detecting pneumocystis jirovecii and drug resistance of pneumocystis jirovecii
  • Kit, reaction system and method for detecting pneumocystis jirovecii and drug resistance of pneumocystis jirovecii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] This embodiment is an example of a kit for detection of Pneumocystis sp. and drug resistance. The kit components of this embodiment are shown in Table 1, and the specification is 25 tests / box.

[0051] Table 1 Pneumocystis and drug resistance detection kit composition

[0052]

[0053]

[0054] The test kit of this embodiment is suitable for the following instruments:

[0055] 480II (Roche)

[0056] Q(QIAGEN)

[0057] CFX96 (Biorad)

[0058] Mic qPCR (Bio Molecular Systems)

[0059] Quantstudio 5 (Thermo Fisher Scientific)

[0060] The storage conditions of the Pneumocystis and Drug Resistance Detection Kit are -15°C to -30°C and protected from light; try to avoid repeated freezing and thawing (no more than 10 times); see the product label for the expiration date. To avoid cross-contamination, it is recommended to operate in a PCR laboratory and store positive controls separately.

[0061] The Mastermix in the kit contains the composition shown in Table...

Embodiment 2

[0065] 1. Nucleic acid extraction

[0066] If the sample is viscous, pretreat the sample with DTT. Add 10% 1M DTT to the samples and incubate at 37°C for 15 minutes. use Blood minikit (QIAGEN) or Extract sample nucleic acid, see instructions for details. Note: Add IC to the sample before nucleic acid extraction.

[0067] Avoid repeated freezing and thawing of the extracted nucleic acid. If it is used on the day of extraction, it is recommended to store the DNA at 4°C, and it can also be stored at -20°C for a long time.

[0068] 2. Operation steps

[0069] 2.1 Preparation of reaction system

[0070] Prepare the PCR reaction system according to Table 3. After the preparation is complete, mix well, dispense 20 μl into PCR reaction wells, and place the reaction plate on ice.

[0071] Table 3 Preparation of Pneumocystis and Drug Resistance Detection Kit Reaction Mixture

[0072] component name Volume (1×) Volume (10×) The reaction solution 10 100 ...

Embodiment 3

[0102] This embodiment is a performance test example of the kit.

[0103] 1. Minimum detection limit

[0104] PCR amplification was performed using a DNA fragment containing mtLSU and DHPS sequences as a template. The template was diluted in a 10-fold gradient with concentrations of 1000copies / μL, 100copies / μL, 10copies / μL, 1copies / μL, and 0.1copies / μL, and the minimum detection limit was determined by the detection rate of ≥95% in 20 repeated tests.

[0105] category LOD(copies / μL) DHPS 1 wxya 1

[0106] 2. Specificity

[0107] In this example, 21 fungal samples of different types were used, and the above-mentioned fungal samples were detected using the kit in Example 1. Only the Pneumocystis sp. samples were tested positive, and the specificity reached 100%.

[0108] fungal type wxya DHPS Pneumocystis + + Cryptococcus neoformans - - Saccharomyces cerevisiae - - Bai Nian - - Candida parapsilosis - ...

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Abstract

The invention discloses a kit, a reaction system and a method for detecting pneumocystis jirovecii and drug resistance thereof. The kit of the invention comprises a first primer, a first probe, a second primer, a second probe, a third primer and a third probe capable of being present in the form of a mixture, wherein the first primer and the first probe are used to detect a first target gene present in multiple copies in a real-time fluorescent PCR first channel; the second primer and the second probe are used for detecting whether a second target gene existing in a single copy mode has drug resistance related mutation or not in a real-time fluorescent PCR second channel; the molar ratio of a forward primer to a reverse primer in the second primer is 1: 15-25, and the Tm value of the second probe is 73-76 DEG C. And the third primer and the third probe are used for detecting internal quality control in a real-time fluorescent PCR third channel. According to the invention, whether the sample has pneumocystis jirovecii or not can be detected, and the drug resistance of the fungus sample to sulfonamides can be analyzed and detected through a melting curve.

Description

technical field [0001] The invention relates to the field of detection of Pneumocystis spp., in particular to a kit, a reaction system and a method for detecting Pneumocystis spp. and its drug resistance. Background technique [0002] Pneumocystis pneumonia (Pneumocystis pneumonia) is a protozoan disease caused by Pneumocystis bacterium (P.jirovecii), which is more common in immunocompromised or deficient persons. Widespread sulfa drug prophylaxis and highly active antiretroviral therapy (HAART) have reduced the incidence of pneumocytosis, but it remains an important cause of morbidity and mortality in HIV / AIDS patients as well as non-HIV immunocompromised patients. [0003] Pneumocystis is diagnosed clinically by direct microscopic examination of bronchoalveolar lavage (BAL) samples, visualized by standard staining techniques and by immunofluorescence. This method is diagnostically difficult and requires a high level of skill when the fungal load in the sample is low. At ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/686C12Q1/689C12Q2600/106C12Q2600/156C12Q2561/113C12Q2563/107
Inventor 夏小凯程天龄朱丽媛吉斯·丁格曼斯
Owner 上海捷诺生物科技有限公司
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