Kit and method for multi-fold fluorescence quantitation detection of cryptococcus neoformans and aspergilli

A multiple fluorescence quantitative and cryptococcus neoformans technology, which is applied in the field of nucleic acid detection, can solve the problems of no dual detection kits for cryptococcus neoformans and aspergillus, and few kits, so as to improve the utilization rate of the instrument, reduce the detection cost, and solve the problem of Effects of Flux Problems

Inactive Publication Date: 2017-10-24
BEIJING FUANHUA BIOLOGICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are very few real-time fluorescent quantitative detection kits for Aspergillus on the market, and almost no kits for dual detection of Cryptococcus neoformans and Aspergillus

Method used

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  • Kit and method for multi-fold fluorescence quantitation detection of cryptococcus neoformans and aspergilli
  • Kit and method for multi-fold fluorescence quantitation detection of cryptococcus neoformans and aspergilli
  • Kit and method for multi-fold fluorescence quantitation detection of cryptococcus neoformans and aspergilli

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1, Multiple real-time fluorescent quantitative detection kits for Cryptococcus neoformans and Aspergillus and special primers thereof

[0048] 1. Design and synthesis of special primers for multiple real-time fluorescence quantitative detection of Cryptococcus neoformans and Aspergillus

[0049] According to the Cryptococcus neoformans IGS-1 gene-specific sequence (sequence 1), the primer sequences were designed as follows:

[0050] The upstream amplification primer sequence of Cryptococcus neoformans is: 5'-GTCTCTAACATGTTGGGTCTCG-3' (SEQ ID NO: 2);

[0051] The downstream amplification primer sequence of Cryptococcus neoformans is 5'-TCCTAGAGTCATCCACACTTCA-3' (SEQ ID NO: 3);

[0052] The sequence of the fluorescent probe for Cryptococcus neoformans is: 5'-CCCTTACATCCAAGTCTCTAGAGGAAGCC-3' (sequence 4), the 5' end is marked with a FAM fluorescent group, and the 3' end is marked with a TAMRA quencher group.

[0053] According to Aspergillus ITS-1 gene specific s...

Embodiment 2

[0091] Example 2, Application of multiple real-time fluorescent quantitative detection kits for Cryptococcus neoformans and Aspergillus

[0092] 1. Sensitivity detection

[0093] Set the concentration to 10 5 copies / ul standard of Cryptococcus neoformans IGS-1 gene sequence and concentration of 10 5 Standard products of the Aspergillus IGS-1 gene sequence of copies / ul are divided by 10 5 、10 4 、10 3 、10 2 , 10copies / ul gradient dilution, and then use it as the sample DNA to be tested respectively, and detect according to the fluorescence quantitative method of the second in the embodiment 1.

[0094] The result is as figure 2 (The curves in the figure are 10 from left to right 5 、10 4 、10 3 、10 2 , 10copies / ul), the upper picture shows Cryptococcus neoformans, and the lower picture shows Aspergillus, it can be seen that the kit and method can detect 10copies / ul of Cryptococcus neoformans DNA and 10copies / ul of Aspergillus DNA copies , with high sensitivity.

[009...

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Abstract

The invention discloses a kit and method for multi-fold fluorescence quantitation detection of cryptococcus neoformans and aspergilli. The invention provides a multi-fold fluorescence quantification reagent for detecting the cryptococcus neoformans and the aspergilli, and the method comprises the following steps of detecting a primer 1 of the cryptococcus neoformans, detecting a primer 2 of the cryptococcus neoformans, detecting a probe 1 of the cryptococcus neoformans, detecting a primer 3 of the aspergilli, detecting a primer 4 of the aspergilli and detecting a probe 2 of the aspergilli. The experiments of the invention prove that the kit and detection method disclosed by the invention are based on multi-fold Taqman fluorescence quantitation detection technology, not only can rapidly make certain of contained pathogens, reduce detection costs and improve the utilization rate of an instrument, but also solve the flux problem of detection of the pathogens contained in clinical respiratory tract fungus, thereby having important guiding significance in early clinical diagnosis and treatment.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a kit and a method for multiplex fluorescent quantitative detection of Cryptococcus neoformans and Aspergillus. Background technique [0002] Invasive fungi include mainly pathogenic fungi such as Candida, Aspergillus, and Cryptococcus, as well as endemic pathogenic fungi such as Histoplasma, Blastomyces dermatitidis, and Sporotrichum. Different environments contain different types of fungi. Invasive fungi have no specific manifestations in the early stage, so they are easily concealed, and the case fatality rate is high. [0003] Cryptococcus neoformans (CN) is a yeast-like pathogenic fungus that can cause severe contamination in humans, mainly causing cryptococcosis in patients with low cellular immunity, such as organ transplantation, lung cancer patients and AIDS patients. [0004] Aspergillus includes 132 species and 18 varieties, the clinically ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645C12R1/66
CPCC12Q1/686C12Q1/6895C12Q2561/113C12Q2563/107C12Q2545/114C12Q2561/101C12Q2537/143
Inventor 乔阳余倩葛猛王宏伟
Owner BEIJING FUANHUA BIOLOGICAL TECH CO LTD
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