Sensitive detection of bacteria by improved nested polymerase chain reaction targeting the 16S ribosomal RNA gene and identification of bacterial species by amplicon sequencing
a technology of ribosomal rna and nested polymerase chain reaction, which is applied in the field of pcr methods, can solve problems such as errors in diagnosis or causation, contamination or experimental artifacts, and inability to detect bacteria, and achieve the effect of improving the detection of such bacterial contaminants
Examples
Embodiment Construction
Material and Methods
Oligonucleotide Primers.
[0031]The oligonucleotide primers used were:
1)Moll Outer Primer (sense)SEQ ID NO: 1(AAYGGGTGAGTAACACGT),2)Gneg Outer Primer (sense)SEQ ID NO: 8(RAYGGGTGAGTAAYGYMT),3)Bact Outer Primer (antisense)SEQ ID NO: 5(CCCGRGAACGTATTCACSG),4)Bact Inner Primer (sense)SEQ ID NO: 6(CTACGGGAGGCWGCAGTRRGGAAT),and5)Bact Inner Primer (antisense)SEQ ID NO: 7(WGGGTATCTAATCCTRTTTGMTCCCCW)[0032]where R=G or A, S=G or C, W=A or T, M=A or C, and Y=T or C.
[0033]Expected lengths of amplicons are ˜1,200 bp and ˜450 bp with the outer and inner primers, respectively. The primers employed were formulated as equal amounts of each primer within the class identified by the sequence.
Nucleic Acid Preparation and PCR / RT-PCR.
[0034]Cell line supernatants (400 μl), human plasma (200-400 μl) and human peripheral blood mononuclear cells (PBMC, 3-10 millions cells) were lyzed with 10 mMTris, pH7.4, 10 mM EDTA, 150 mM NaCl, 0.4% SDS, and 10 μg Proteinase K at 60° C. for 1 h. Nuclei...
PUM
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Description
Claims
Application Information
- IPC
- C12Q1/68
- CPC
- C12Q1/689
- Inventors
- MONTAGNIER, LUC; LAVALLEE, CLAUDE