Detection of rare microbiological nucleic acids
A microbial and rare technology, applied in the field of detection of rare microbial nucleic acids, can solve the problems of insufficient hydrolysis steps and inaccessibility
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Embodiment 1
[0147] Example 1: Assessing the number of reads required for detection
[0148] Table 1 summarizes the number of reads that need to be generated to detect virus particles from PCV1 (the smallest known viral genome) and PRV (representing the larger viral genome size) in samples prepared from contaminated hosts . Table 1 is based on the assumption that the viral genome is present in samples containing 1 μg or 1 ng of total nucleic acid molecules and that the sequences are consistently amplified when using the NGS method.
[0149] Table 1
[0150]
[0151] The number of reads was dependent on the size of the viral genome, with small genome sizes increasing the number of reads by 2log10 compared to larger genome sizes. Even though the capacity of NGS instruments is improving, the total number of reads is very important to the performance of the instrument.
Embodiment 2
[0152] Example 2: Effect of DSN Treatment on Amplification of Rare Nucleic Acids
[0153] Supernatants from human MRC5 cells were obtained and artificially spiked with viruses with different types of genomes at virus concentrations close to the detection limit of qPCR (corresponding to the LOD of quantitative (RT)-PCR). Spike the following viruses: human adenovirus type 5 (HADV-5 or Adeno 5, or Ade5; produced and titrated in A549 cells), parvovirus B19 (B19, World Health Organization library, NIBSC 99 / 800, titers in international expressed in units), human polyomavirus BK (BK ATCC-837), NADL strain of bovine viral diarrhea virus (BVDV-1-NADL or BVDV, produced and titrated in MDBK cells), hepatitis B virus (HBV, WHO library, NIBSC97 / 750, titers in IU), equine influenza virus A (InfA or FluA H3N8, produced in MDCK cells and titrated in genome copy number (gc) per ml by quantitative PCR), Moloney murine leukemia Viruses (MoMLV ATCC VR-190, produced in NIH / 3T3 and titrated in Fgl...
Embodiment 3
[0205] Example 3: Comparing the Sensitivity of Detection by qPCR, Detection by NGS and Detection by the Method of the Invention in the Methods of the Prior Art
[0206] As described in Example 1, MRC5 supernatants were artificially spiked with 9 different viruses at concentrations close to the qPCR detection limit of the viruses. Two series of samples were prepared for each virus:
[0207] - 0.8x LOD concentration of virus (mixture 1)
[0208] - 3x LOD concentration of virus (mixture 2)
[0209] Virus dilutions were consistent with those previously published in the NGS method (NGS method 1) described in Cheval et al. sensitivity. Table 3 below summarizes the virus names and references.
[0210] table 3
[0211]
[0212] · NGS method 1 (Cheval et al. 2011)
[0213] A volume of 150 μl of each sample was extracted using the nucleospin RNA virus kit (Macherey-Nagel) and subsequently amplified by multiple displacement amplification (MDA) based on bacteriophage Phi29 polyme...
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