Assays for resistance to echinocandin-class drugs

A technology of echinocandin and detection method, which is applied in the field of fungal nucleic acid detection method, can solve the problems of decreased sensitivity of candida and low inhibitory concentration value, etc.

Active Publication Date: 2009-06-03
UNIV OF MEDICINE & DENTISTRY OF NEW JERSEY (US) +1
View PDF6 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the rapid increase in the use of caspofungin, clinical isolates of Candida have been reported to be less sensitive in vitro, and there is a close correlation between treatment failure and higher minimum inhibitory concentration or MIC in vitro

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Assays for resistance to echinocandin-class drugs
  • Assays for resistance to echinocandin-class drugs
  • Assays for resistance to echinocandin-class drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Nucleic acid amplification of relevant fragments of the CaFKS1 gene and cycle sequencing of identifying mutants used in conjunction with it have been successfully performed using 4 different strains. A CaFKS1 (approximately 450 bp) fragment was amplified from genomic DNA of strains CAI4-R1, NR2, NR3 and NR4. Based on the sequence of CaFKS1 (GenBank Accession No. D88815), the sense and antisense primers used in PCR were 5'-GAAATCGGCATATGCTGTGTC-3' and 5'-AATGAACGACCAATGGAGAAG-3', respectively. The PCR product was cloned into pCR2.1 (Invitrogen) and the DNA sequence was determined. For Candida clinical isolates, 5′-CATTGCTGTGGCCACTTTAG-3′ and 5′-GGTCAAATCAGTGAAAACCG-3′ were used as forward and reverse primers, respectively, to amplify most of the CaFKS1 ORF (about 2.6 kb) for DNA sequence analysis. In addition to the above-mentioned first target region of CaFKS1 (consistent with 1921st to 1947th coding nucleotides), this fragment includes the second target nucleotides fr...

Embodiment 2

[0063] DNA sequence analysis using nucleic acid amplification and cycle sequencing methods can be used as a stand-alone detection technique or as a control to evaluate probe-based assays.

[0064] C. albicans chromosomal DNA was extracted from cells grown overnight in liquid YPD medium using the Q-Biogene FastDNA kit (Q-Biogene, Irvine, CA). PCR experiments were performed on an iCyclerthermocycler (Bio-Rad Laboratories, Hercules, CA). Using primers CaFKS1-F1719 and CaFKS1-R2212 ( Figure 4 ) amplifies the region of CaFKS1 designated HS1. Each 100-μl PCR reaction system contained two primers at 0.25 μM, 2.5 U of iTaq DNA polymerase (Bio-Rad Laboratories, Hercules, CA), 0.5 mM dNTPs, 50 mM KCl, 4 mM MgCl 2 , 20mM Tris-HCl, pH=8.4, about 50ng of Candida albicans chromosomal DNA. The cycle conditions were 1 cycle, 95°C for 3 minutes; 35 cycles, 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute; 1 cycle, 72°C for 3 minutes. PCR products were purified using Montage...

Embodiment 3

[0068] It has been reported that three CaFKS1 DNA sequences with GenBank accession numbers D88815, AF027295 and CA2043 were used for FKS1 molecular beacon and primer design. The design of primers and probes for detection is known in the art. A number of published documents are available to assist the researcher. Additional computer software packages are available to increase efficiency and reduce the need for trial-and-error adjustments (see Example 4). We use this package. Molecular beacons and DNA primers ( Figure 4 ). Preset software parameters are suitable for the construction of all molecular beacons and primers. With the fluorophores 5-carboxyfluorescein (FAM) and 6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein (HEX) at the 5′ end and dabcyl at the 3′ end Label molecular beacons. Molecular beacons and primers were purchased from Biosearch Technologies (Biosearch Technologies, Novato, CA). The hybridization properties of CaFKS1 allele-specific molecular beacons hy...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention provides nucleic acid amplification assays for mutations to two short sections of the fungal gene FKS1. Mutations in these target sequences have been shown to correlate with resistance to echinocandin-class drugs. Assays may include detection by sequencing or by labeled hybridization probes. Also, primers, probes and reagent kits for performing such assays.

Description

technical field [0001] The invention relates to a nucleic acid detection method for fungi. Background of the invention [0002] Fungal infection is an important cause of morbidity and death in critically ill patients, and the consequences of infection are aggravated due to the failure to diagnose quickly and treat effectively. The widespread use of antifungal drugs has led to a selection effect on naturally resistant fungal species and the emergence of resistance in susceptible species. The limited range of effective antifungal drugs hinders the treatment of fungal diseases. Recently, caspofungin, as the first new echinocandin drug, was used clinically. This kind of drug blocks β-(1→3)-D-glucan synthase and acts on the fungal cell wall. With the rapid increase in the use of caspofungin, it has been reported that the sensitivity of clinical isolates of Candida in vitro is reduced, and treatment failure is closely related to the minimum inhibitory concentration value or MIC ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C07H21/00C07H19/00
CPCC12Q1/6827C12Q1/6895C12Q2600/16C12Q2535/131C12Q2531/113C12Q1/6844C12Q1/6876C12Q2600/156
Inventor D·S·珀林S·帕克C·M·杜格拉斯J·N·卡恩S·A·帕伦特R·凯利
Owner UNIV OF MEDICINE & DENTISTRY OF NEW JERSEY (US)
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap