Compositions and methods for rapid identification of bacteria and fungi and phenotypic antimicrobial drug sensitivity assays

A drug sensitivity and antimicrobial technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc.

Pending Publication Date: 2022-07-08
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Disk diffusion method provides only qualitative results

Method used

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  • Compositions and methods for rapid identification of bacteria and fungi and phenotypic antimicrobial drug sensitivity assays
  • Compositions and methods for rapid identification of bacteria and fungi and phenotypic antimicrobial drug sensitivity assays
  • Compositions and methods for rapid identification of bacteria and fungi and phenotypic antimicrobial drug sensitivity assays

Examples

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example

[0226] The following examples, tables, and figures are provided to assist in understanding the subject matter, the true scope of which is set forth in the appended claims. It should be understood that modifications to the illustrated procedures may be made without departing from the spirit of the invention.

example 1

[0227] Example 1 PCR conditions

[0228] use Real-time PCR detection of target genes was performed on the 6800 / 8800 system platform (Roche Molecular Systems, Inc., Pleasanton, CA). The final concentrations of amplification reagents are as follows:

[0229] Table XXIX: PCR amplification reagents

[0230]

[0231] The following table shows a typical temperature profile for a PCR amplification reaction:

[0232] Table XXX: PCR Temperature Profile

[0233]

[0234] The Pre-PCR procedure included initial denaturation and incubations at 55°C, 60°C and 65°C for reverse transcription of the RNA template. Incubation at the three temperatures simultaneously has the following beneficial effects: at lower temperatures, slightly mismatched target sequences (such as genetic variants of an organism) are also transcribed, while at higher temperatures, changes in RNA secondary structure Formation is inhibited, thereby making transcription more efficient. The PCR cycle was divide...

example 2

[0235] Example 2 PCR using primers / probes of interest for the detection of Enterobacter

[0236]Using the PCR conditions described in Example 1, forward primer RM_ENTF (SEQ ID NO: 1), reverse primer RM_ENTRP (SEQ ID NO: 2) targeting the rplP gene, and two probes RM_ETP02 (SEQ ID NO: 2) were used 3) and RM_ETP02B (SEQ ID NO: 4) PCR assay was performed on the following bacterial strains: five bacterial strains in the order Enterobacteriaceae: Escherichia coli (E.Coli), K. pneumoniae (K.pneumonia), Semen Enterobacter (E.cloacae), Serratia marcescens (S.marcescens), Proteus mirabilis (P.mirabilis); and two bacterial strains from non-Enterobacteriaceae: Pseudomonas aeruginosa (P.aeruginosa) and Acinetobacter baumannii (A. baumannii). For cultures of all strains (overnight cultures previously stored in glycerol), concentrations of starting material were used in the range between 1e8 CFU / ml and 5e8 CFU / ml with DNA (approximately 1e7 copies / μl) ) except S. marcescens. No sample p...

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Abstract

The present invention relates to compositions and methods for using polymerase chain reaction (PCR) as a reporter assay for rapid and simultaneous bacterial identification and antimicrobial drug sensitivity (AST) phenotypic assays. The present invention uses a strategy that has exhibited the ability for multi-reuse as well as processing of a variety of microbiological samples for antimicrobial drug sensitivity tests.

Description

technical field [0001] The present disclosure relates to the field of molecular diagnostics, and more particularly to the identification and determination of antimicrobial susceptibility of bacteria from biological samples. Background technique [0002] There is an urgent need to develop methods for the rapid and easy detection, identification, and determination of antimicrobial susceptibility of bacterial pathogens in clinical samples to guide the diagnosis and treatment of infectious diseases. A good example of this need is in the field of blood infection (BSI). BSI ranks among the top seven causes of death in North America and Europe, and it is estimated that 1.7 million sepsis events occur in the United States each year, resulting in 270,000 deaths and $14 billion in annual U.S. health care costs. Shortening the time to directed antimicrobial therapy has been shown to improve morbidity and mortality in sepsis patients, resulting in shorter length of stay (LOS) and lower...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/689C12Q1/686C12Q2561/101C12Q2561/113C12Q2537/143C12Q2563/107C12Q1/18C12Q1/6851C12Q1/6895C12Q1/6897C12Q2565/1015C12Q2600/136
Inventor P·阿邦K·C·卡迪R·陈B·汉森R·梅塔T·拉邦P·林
Owner F HOFFMANN LA ROCHE & CO AG
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