Real-time fluorescence RPA detection kit for aspergillus fumigatus as well as special primer and probe thereof

A kit, the technology of Aspergillus fumigatus, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of expensive equipment, false negatives, difficult clinical detection of Aspergillus fumigatus, etc.

Active Publication Date: 2020-02-07
中国人民解放军疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the patient's own low immunity and antifungal treatment, false negative or false positive results may be produced by serological testing [SuLahian A, Touratier S, Ribaud P. False positive test for aspergillus antigenemia related to concomitant administration of piperacillin and tazobactam. N Engl J Med.2003,349:2366-2367.]
The detection method of molecular biology mainly detects the specific genes of Aspergillus fu

Method used

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  • Real-time fluorescence RPA detection kit for aspergillus fumigatus as well as special primer and probe thereof
  • Real-time fluorescence RPA detection kit for aspergillus fumigatus as well as special primer and probe thereof
  • Real-time fluorescence RPA detection kit for aspergillus fumigatus as well as special primer and probe thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1 designs the real-time fluorescent RPA primer that Aspergillus fumigatus detects and exo probe

[0068] The sequence (GenBank: KU575861.1) of the annexin anxC4 gene (anxC4 or ANXC4) of Aspergillus fumigatus was retrieved from NCBI's nucleic acid database GenBank (http: / / www.ncbi.nlm.nih.gov), and performed with DNA Man software. After comparison, the Aspergillus fumigatus anxC4 gene was obtained as the specific conserved sequence of Aspergillus fumigatus (it is specific to other fungi and humans, so it is used as a detection template); select the Aspergillus fumigatus anxC4 gene (GenBank: KU575861.1) The reverse complementary sequence is used as a template, and the primers and For exo probe design, a total of 1 probe and multiple upstream primers and downstream primers are designed. Any 1 upstream primer and 1 downstream primer can form a set of combinations. A set of primer-probe combinations with the best performance was screened through sensitivity te...

Embodiment 2

[0078] Embodiment 2 uses primer of the present invention and Exo probes for real-time fluorescent RPA detection of Aspergillus fumigatus

[0079] 1. Extract the genomic DNA of Aspergillus fumigatus

[0080] Extract the genomic DNA of Aspergillus fumigatus (ATCC13073) with CTAB method, and concrete method comprises the following steps:

[0081] 1) Prepare CTAB (cetyltrimethylammonium bromide) extraction buffer (pH 8.0): weigh 30g of cetyltrimethylammonium bromide (CTAB), 12.12g of Tris, ethylenediaminetetra Acetic acid (EDTA) 5.84g, sodium chloride (NaCl) 82g, fully stirred and dissolved with 800mL deionized water, then add 100mL dithiothreitol (DTT) solution, adjust pH to 8.0 with NaOH, and finally dilute to 1L, Store at room temperature;

[0082] 2) Prepare CIA solution: CIA saturated phenol / chloroform / isoamyl alcohol 25:24:1 (V / V / V), store at 4°C;

[0083] 3) Add 600 μL of CTAB extraction buffer to the Aspergillus fumigatus sample, shake and mix well, and lyse at 65°C (...

Embodiment 3

[0100] Embodiment 3 detects the specificity of Aspergillus fumigatus real-time fluorescence RPA detection method

[0101] Aspergillus fumigatus (ATCC 13073), Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, Aspergillus versicolor, Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida glabrata, Cryptomonas Coccus, Cryptococcus nails, Cryptococcus neoformans, Penicillium citrinum, Penicillium manefei, Exophiala acanthus, Exophiala dermatitidis, Histoplasma capsulatus, Fusarium oxysporum, Trichophyton rubrum, Golden yellow Staphylococcus, Acinetobacter baumannii (the above strains are all from the Nosocomial Infection Supervision Center of the Chinese People's Liberation Army Institute for Disease Control and Prevention), human A549 cell gene DNA as templates, and 10 4 Copy / 2μL Aspergillus fumigatus cloning plasmid pMD19-T-anxC4 standard product was used as a positive control, and sterile deionized water was used as a...

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Abstract

The invention relates to a real-time fluorescence RPA detection kit for aspergillus fumigatus as well as a special primer and a probe of thereof, and particularly provides a primer composition for RPAdetection, the primer composition comprises a forward primer and a reverse primer, the forward primer has a nucleotide sequence shown as SEQ ID NO: 1, and the reverse primer has a nucleotide sequenceshown as SEQ ID NO: 2. The primer composition for RPA detection can be used for rapidly, conveniently, efficiently, specifically and sensitively detecting aspergillus fumigatus components in samplesto-be-detected, such as thalli, sputum, blood and bronchoalveolar lavage fluid, under an isothermal condition.

Description

technical field [0001] The invention relates to the field of biological detection, in particular, the invention relates to a real-time fluorescent RPA detection kit for Aspergillus fumigatus and special primers and probes thereof. Background technique [0002] Aspergillus fumigatus is a common opportunistic pathogen that is widely distributed in the environment. Its spores can easily cause invasive aspergillosis after being inhaled by immunocompromised or immunocompromised patients. Because its pathogenic mechanism is still unclear, There is no effective treatment, and the fatality rate is as high as 90%. The main fungus that causes invasive aspergillosis is Aspergillus fumigatus. Generally, the infection of Aspergillus fumigatus can only be detected in the late stage of the disease or after death. Therefore, early diagnosis and prevention of Aspergillus fumigatus is very important. In the past few decades, with the high-intensity immunosuppressive treatment and the widespr...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/68
CPCC12Q1/6895C12Q1/6844C12Q2521/507C12Q2522/101C12Q2531/119
Inventor 田曙光韩黎陈芳艳苏雪婷胡颖嵩陈勇赵静雅
Owner 中国人民解放军疾病预防控制中心
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