Methods and kits to identify
The present invention provides a method of detecting one or more Klebsiella species within a sample from a subject, the method comprising: subjecting DNA and/or RNA from the sample to a PCR amplification reaction using primer pairs targeting species-specific canonical single nucleotide polymorphisms (canSNPs); and analyzing amplification products resulting from the PCR amplification reaction to detect the one or more Klebsiella species. The present invention also provides a kit for detection of one or more Klebsiella species, Klebsiella clonal groups, AMR genes, and/or virulence genes, the kit comprising primer pairs targeting species-specific canSNPs, K. pneumoniae genes M1 and M2, clonal group-specific canSNPs, AMR genes, and/or virulence genes.
1. A method of detecting one or more Klebsiella species within a sample from a subject, the method comprising:
selecting a primer pair, wherein each primer of the primer pair comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5-10;
subjecting DNA, RNA, or both from the sample to a PCR amplification reaction using the primer pair; and
detecting the Klebsiella species by detecting amplification products resulting from the PCR amplification reaction.
2. The method of claim 1 , wherein the one or more Klebsiella species are selected from the group consisting of: K. quasipneumoniae, K. oxytoca , and K. variicola.
3. The method of claim 1 , further comprising:
selecting a second primer pair from the group consisting of: primers with a nucleic acid sequence comprising a sequence set forth in SEQ ID NOs: 11-84;
subjecting the DNA, RNA, or both from the sample to a PCR amplification reaction using the second primer pair, and
detecting the Klebsiella clonal group by detecting amplification products resulting from the PCR amplification reaction, wherein the amplification products resulting from the PCR amplification comprises a Klebsiella clonal group-specific canSNP.
4. The method of claim 3 , wherein the amplification products resulting from the PCR amplification using the primer pair comprises the clonal group-specific canSNP selected from the group consisting of: CG258 and its subgroups, CG20 and its subgroup ST20, ST23, ST380, CG37, and CG14 and its subgroups.
5. The method of claim 4 , wherein each primer of the primer pair comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 13-26, 37-38, 63-78, and 81-84.
6. The method of claim 1 , further comprising:
selecting a primer pair targeting an antimicrobial resistance (AMR) gene in the Klebsiella species, wherein each primer of the primer pair targeting an AMR gene comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 85-248;
subjecting the DNA, RNA, or both from the sample to a PCR amplification reaction using a primer pair targeting the AMR gene, and
detecting the AMR gene.
7. The method of claim 6 , wherein the primer pair targets a beta-lactamase gene, a colistin resistance gene, or both.
8. The method of claim 7 , wherein the colistin resistance gene is mcr-1.
9. The method of claim 7 , wherein each primer of the primer pair targeting the beta-lactamase gene comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 85-117, and the primer pair targeting the colistin resistance gene contains sequences selected from the group consisting of SEQ ID NOs: 236-237.
10. The method of claim 1 , further comprising:
selecting a primer pair targeting a virulence gene in the Klebsiella species, wherein each primer of the primer pair targeting the virulence gene in the Klebsiella species comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 249-281;
subjecting the DNA, RNA, or both from the sample to a PCR amplification reaction using the primer pair targeting the virulence gene, and
detecting the virulence gene.
11. The method of claim 10 , wherein the primer pair targets rmpA, wzi, or both, each primer of the primer pair targeting rmpA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 249-250, and each pair of the primer pair targeting wzi comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 280-281.
12. The method of claim 1 , wherein the PCR amplification reaction is a multiplex amplification reaction.
13. The method of claim 1 , wherein the amplification products of PCR amplification reaction are detected by next-generation sequencing (NGS).
14. The method of claim 1 , wherein the subject has pneumonia, the method further comprises treating the subject with an aminoglycoside, cephalosporin, or both upon detection of the Klebsiella species.
15. A composition for detecting one or more Klebsiella species comprising:
a primer pair, wherein the nucleic acid sequence of each primer of the primer pair comprises a sequence selected from the group consisting of: SEQ ID NOs: 5-10; and a universal tail sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 282-283; and
a nucleotide polymerase, buffer, diluent, excipient, or combinations thereof.
16. The composition of claim 15 , further comprising a primer pair for detecting a Klebsiella clonal group, wherein the nucleic acid sequence of each primer of the primer pair for detecting the Klebsiella clonal group is selected from the group consisting of: SEQ ID NOs: 11-84.
17. The composition of claim 15 , further comprising a primer pair for detecting an AMR gene in the Klebsiella species, wherein the nucleic acid sequence of each primer of the primer pair for detecting the AMR gene in the Klebsiella species is selected from the group consisting of: SEQ ID NOs: 85-248.
18. The composition of claim 15 , further comprising a primer pair for detecting a virulence gene in the Klebsiella species, wherein the nucleic acid sequence of each primer of the primer pair for detecting the virulence gene in the Klebsiella species is selected from the group consisting of: SEQ ID NOs: 249-281.
19. A composition for detecting one or more Klebsiella species comprising a primer pair, wherein the nucleic acid sequence of each primer of the primer pair comprises a sequence selected from the group consisting of: SEQ ID NOs: 5-10; and a universal tail sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 282-283.