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Application of Aspergillus fumigatus annexin anxC4 gene (anxC4 gene)

A technology of Aspergillus fumigatus film and Aspergillus fumigatus, which is applied in the field of biotechnology, and can solve the problems of difficult rapid clinical detection of Aspergillus fumigatus, expensive instruments and equipment, false negatives, etc., and achieves simple result judgment method, short detection time and high specificity Effect

Inactive Publication Date: 2016-06-01
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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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, the immunological detection method may produce false negative or false positive results [SulahianA, TouratierS, RibaudP.
The detection method of molecular biology mainly detects the specific genes of Aspergillus fumigatus through PCR technology (including fluorescent quantitative PCR). PCR technology has the advantages of high specificity, accuracy, sensitivity, and rapidity. The requirements are very high, the equipment is expensive, and strict temperature control is required, so it is difficult to apply to the clinical rapid detection of Aspergillus fumigatus [Ostrosky-ZeichnerL.Invasivemycoses: diagnostic challenges.AmJMed.2012,125:S14-24.]

Method used

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  • Application of Aspergillus fumigatus annexin anxC4 gene (anxC4 gene)
  • Application of Aspergillus fumigatus annexin anxC4 gene (anxC4 gene)
  • Application of Aspergillus fumigatus annexin anxC4 gene (anxC4 gene)

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Experimental program
Comparison scheme
Effect test

Embodiment 1-1

[0074] Embodiment 1-1, design the LAMP detection primer of Aspergillus fumigatus

[0075] The sequence of the anxC4 gene (anxC4) of Aspergillus fumigatus (A. fumigatus) was retrieved from the NCBI nucleic acid database GenBank (http: / / www.ncbi.nlm.nih.gov) (GenBank: AY598940.1), through BLAST software Carry out homology analysis, obtain Aspergillus fumigatus annexin anxC4 gene is the specific conserved sequence of Aspergillus fumigatus (SEQ ID NO: 1 in the sequence table, is the full length of anxC4 gene, it is specific relative to other fungi and people, therefore as detection template), and then according to the specific conserved sequence (SEQIDNO: 1), use the software PrimerExplorerversion4 (http: / / primerexplorer.jp / e) to design primer combinations for LAMP detection of Aspergillus fumigatus, and design more than 30 sets of primers Combinations (parts of which are shown in Table 2), a set of primers with the best performance were screened out through sensitivity and specif...

Embodiment 1-2

[0081] Embodiment 1-2, establish the LAMP detection method of Aspergillus fumigatus

[0082] 1) Construction of a cloning plasmid (standard product) carrying the annexin ANXC4 gene (anxC4) of Aspergillus fumigatus (A.fumigatus)

[0083] According to the nucleotide sequence (GenBank: AY598940.1) of Aspergillus fumigatus (A.fumigatus) annexin anxC4 gene (anxC4), the primers for PCR amplification of Aspergillus fumigatus anxC4 gene (upstream primer F sequence: 5'-TCGCCAAATCTTCGACCAGT-3 ', downstream primer B sequence: 5'-GGTCTGGCATAGCTGGGATG-3'), with the improved cetyltrimethylammonium bromide method (CTAB) (AttitallaIH.ModifiedCTABmethodforhighqualitygenomicDNAextractionfrommedicinalplants.PakJBiolSci.2011Nov1;14(21):998- 9.) Extract the genomic DNA of Aspergillus fumigatus (ATCC13073), then use the genomic DNA of Aspergillus fumigatus as a template, and perform PCR amplification under the guidance of primers F and B. The PCR reaction system is 50 μL (10 × PCR reaction buffer 5...

Embodiment 1-3

[0098] Embodiment 1-3, the specific detection of the LAMP detection method of Aspergillus fumigatus

[0099] Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, Aspergillus versicolor, Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida glabrata, Cryptococcus albicans, nail Cryptococcus, Cryptococcus neoformans, Penicillium citrinum, Penicillium manefei, Exophiala spinosa, Exophiala dermatitidis, Histoplasma capsulatus, Fusarium oxysporum, Trichophyton rubrum, Staphylococcus aureus, Acinetobacter baumannii (the above strains are all from the Infectious Disease Control Center of the Chinese People's Liberation Army's Center for Disease Control and Prevention) and the genomic DNA of human lung adenocarcinoma cell A549 (Human) were used as templates to carry the Aspergillus fumigatus annexin ANXC4 gene (anxC4) The cloning plasmid is standard, with sterile deionized water as a negative control, th...

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Abstract

The invention discloses application of Aspergillus fumigatus annexin anxC4 gene (anxC4 gene). The anxC4 gene can be used as an Aspergillus fumigatus detection target. The invention also discloses a LAMP (loop-mediated isothermal amplification) kit designed according to the specific conserved sequence of the anxC4 gene and special primers thereof, and an Aspergillus fumigatus real-time fluorescent quantitative PCR (polymerase chain reaction) detection kit and primers and TaqMan probes thereof. The technique can be used for qualitatively detecting Aspergillus fumigatus components in phlegm, blood, bronchus alveoli douche and other samples to be detected, and can be used for quickly and conveniently detecting Aspergillus fumigatus under isothermal conditions in a high-efficiency high-specificity high-sensitivity way.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to the use of Aspergillus fumigatus annexin anxC4 and its gene (anxC4 gene) as a target for detection of Aspergillus fumigatus, and relates to the application of the anxC4 gene as a detection target for nucleic acid detection, immune detection, and biochemical detection, and the application The Aspergillus fumigatus LAMP kit and its primers, the fluorescent quantitative PCR detection kit and its primers and TaqMan probes obtained in . Background technique [0002] Current status of clinical infection and detection of Aspergillus fumigatus [0003] 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. Effective treatment, the case fatality rate is as high as 90%. The main fungus that causes invasive aspergillosis is Asper...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N33/68C12R1/68
CPCC12Q1/6895C12Q1/6844C12Q1/6851G01N33/6854
Inventor 韩黎田曙光唐青韩雪琳陈勇赵静雅陈芳艳
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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