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Microbial markers and uses therefor

A mammalian, bacterial technology, applied in the field of microbial markers and their uses, which can solve problems such as lack of detection

Inactive Publication Date: 2016-04-13
IMMUNEXPRESS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another technical difficulty associated with PCR-based pathogen detection, especially in peripheral blood samples, is the lack of ability to detect small amounts of pathogen nucleic acid in the background of host nucleic acid

Method used

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  • Microbial markers and uses therefor
  • Microbial markers and uses therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0392] Identification of 16S rRNA SNPs that distinguish between Gram-negative and Gram-positive prokaryotes

[0393] Representative genes encoding 16S rRNA molecules were downloaded from GenBank and aligned using CLUSTALW to determine conserved sequence regions. Variable sequences as determined by the CLUSTALW alignment were removed, and the sequences were re-aligned using CLUSTALW and checked for any additional variable regions. This process is repeated several times. Subsequently, a final conserved mega-alignment of all genes encoding 16S rRNA was generated, consisting of a conserved sequence region of approximately 702 base pairs. The gene encoding an exemplary 16S rRNA from E. coli (Genbank Accession No. NR_074891) is listed in SEQ ID NO: 1, and a 702 base pair conserved region extends from nucleotides 254-955 of SEQ ID NO: 1.

[0394] The aligned sequences were analyzed and it was determined that two SNPs were sufficient to distinguish most Gram-positive and Gram-negati...

Embodiment 2

[0398] In silico discrimination of Gram-negative and Gram-positive prokaryotes using SNP396 and SNP398

[0399] In silico analysis was used to evaluate which prokaryotes could utilize only SNP396 and 398 and be classified on the basis of their Gram status. Twelve (12) base pair probes (GC(A / C / G / T)A(A / C / G / T)G(CC / TA)GCGT; SEQ ID NO: 2) were used in BLAST searches To identify the region of prokaryotic 16SrRNA spanning positions 396 and 398 (numbering corresponding to the Escherichia coli 16SrRNA listed in SEQ ID NO: 1), and analyze the results to determine which species can be correctly classified as Gram-positive or Gram-positive based on these SNPs. Gram negative.

[0400] Table 31 lists the most common mammalian pathogens that were typed as Gram-negative or Gram-positive based only on SNP positions 396 and 398. Most pathogens can be typed as Gram-negative or Gram-positive based on these two SNPs, except for some Gram-negative bacteria that have A and C or G and C at SNP posi...

Embodiment 3

[0426] In silico Discrimination of Gram-negative and Gram-positive sepsis-associated bacteria using SNPs

[0427] As shown in Example 2 (Table 31), there are several instances where the Gram status of a mammalian (eg, human) pathogen cannot be determined using the SNP at positions 396 and 398. These pathogens include the following Gram-negative genera: Helicobacter, Veillonella, some Bacteroides, Campylobacter, and Chlamydia tropophilia. Although the most common mammalian (eg, human) sepsis-associated bacteria (eg, S. aureus, S. epidermidis, S. hemolyticus, S. hominis, S. saprophyticus, E. faecalis, E. faecium, Clostridium perfringens, Streptococcus viridans group (Streptococcus angina, Streptococcus constellation, Streptococcus intermedius, Streptococcus mitis, Streptococcus mutans, Streptococcus sanguis, Streptococcus aureus, and Streptococcus oralis), Streptococcus pneumoniae , Streptococcus agalactiae, Streptococcus bovis, Streptococcus sanguis, Streptococcus dysgalactiae...

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Abstract

Disclosed are methods for identifying and / or classifying microbes using one or more single nucleotide polymorphisms (SNPs) in 16S ribosomal RNA (16S rRNA) of prokaryotes and / or one or more SNPs in 5.8S ribosomal RNA (5.8S rRNA) of eukaryotes. Also disclosed are probes, primers and kits that are useful in those methods. Methods for the diagnosis of sepsis based upon these SNPs are also disclosed.

Description

[0001] related application [0002] This application claims priority to Australian Provisional Application No. 2013901907, filed 28 May 2013, entitled "Microbial Markers and Uses Therefor" and Australian Provisional Application No. 2013903914, filed 11 October 2013, entitled "Microbial Markers and Uses Therefor", each of which The subject matter is incorporated herein by reference in its entirety. field of invention [0003] The present invention generally relates to the use of one or more single nucleotide polymorphisms (SNPs) in the 16S ribosomal RNA (16SrRNA) of prokaryotes and / or the 5.8S ribosomal RNA (5.8SrRNA) of eukaryotes A method of identifying and / or classifying microorganisms by one or more SNPs in . The invention also relates to probes, primers and kits useful in the methods of the invention. Background of the invention [0004] Prokaryotes are organisms that lack a nucleus or any membrane-bound organelles and are generally unicellular. While most prokaryotes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G16B30/10G16B35/00
CPCC12Q1/689C12Q2600/156G16B30/00G16B35/00G16C20/60A61P31/04G16B30/10C12Q1/6895
Inventor 理查德·布鲁斯·布兰登弗拉维娅·惠更斯
Owner IMMUNEXPRESS
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