Multiplex amplification reaction method for determination of Campylobacter jejuni Penner/capsule type

Patent metadata

Field	Value
Publication number	US-9328389-B2
Application number	US-201313963146-A
Country	US
Kind code	B2
Filing date	Aug 9, 2013
Priority date	Feb 24, 2010
Publication date	May 3, 2016
Grant date	May 3, 2016

Abstract

The inventive method and associated reagents relate to a molecular approach to determining Campylobacter jejuni capsule/Penner types. The invention also relates to a method of identifying Campylobacter jejuni types using primers in a multiplex PCR assay.

First claim

What is claimed is:

1. A method of identifying Campylobacter jejuni strains in a sample suspected of containing Campylobacter jejuni DNA by polymerase chain reaction, wherein the amplification products of said polymerase chain reaction are derived from genes within the Campylobacter jejuni polysaccharide capsule (CPS) loci, comprising: (a) subjecting DNA from said sample to a PCR amplification reaction using one or more PCR primer pairs targeting one or more regions of the C. jejuni O-methyl phosphoramidate synthesis region, heptose synthesis and hyper-variable region of the Campylobacter jejuni polysaccharide capsule loci; (b) analyzing amplification products resulting from said amplification reaction, wherein said polysaccharide capsule loci is derived from Campylobacter jejuni strains selected from the group consisting of HS19, HS63, HS33, HS35, HS57, HS12, HS27, HS21, HS31, HS62, HS45, HS29, HS22, HS9, HS37, HS18, HS58, HS52, HS60, HS55, HS32, HS11, HS40, and HS38.

2. The method of claim 1 , wherein said amplification products are analyzed by size determination.

3. The method of claim 1 , wherein said PCR primer pairs contain sequences selected from the group consisting of: SEQ ID No. 1 and SEQ ID No. 2; SEQ ID No. 3 and SEQ ID No. 4; SEQ ID No. 5 and SEQ ID No. 6; SEQ ID No. 7 and SEQ ID No. 8; SEQ ID No. 9 and SEQ ID No. 10; SEQ ID No. 11 and SEQ ID No. 12; SEQ ID No. 13 and SEQ ID No. 14; SEQ ID No. 15 and SEQ ID No. 16; SEQ ID No. 17 and SEQ ID No. 18; SEQ ID No. 19 and SEQ ID No. 20; SEQ ID No. 21 and SEQ ID No. 22; SEQ ID No. 23 and SEQ ID No. 24; SEQ ID No. 25 and SEQ ID No. 26; SEQ ID No. 27 and SEQ ID No. 28; SEQ ID No.29 and SEQ ID No. 30; SEQ ID No. 31 and SEQ ID No. 32; SEQ ID No. 33 and SEQ ID No. 34; SEQ ID No. 35 and SEQ ID No. 36; SEQ ID No. 37 and SEQ ID No. 38; SEQ ID No. 39 and SEQ ID No. 40; SEQ ID No. 41and SEQ ID No. 42; SEQ ID No. 43 and SEQ ID No. 44; and SEQ ID No. 45 and SEQ ID No. 46.

4. The method of claim 1 , wherein said PCR reaction is multiplex amplification reaction.

5. The method of claim 1 , wherein said primers are grouped in an alpha mix and a beta mix with the alpha and beta mixes that are separately added to an unknown DNA sample in order to discriminate product sizes.

6. The method of claim 1 , wherein said sample is a clinical sample.

7. The method of claim 1 , wherein said sample is collected from a matrix selected from the group consisting of a bacterial culture, a blood, a tissue, and fecal material.

8. The method of claim 1 , wherein the primers have about 18-30 nucleotides, a G/C content of 20-50%, and a melting temperature between about 57° C. and 63° C.

9. The method of claim 1 , wherein said amplification reaction yields one or more amplification products selected from the group consisting of SEQ ID No. 47;
SEQ ID No. 48; SEQ ID No. 49; SEQ ID No. 50; SEQ ID No. 51; SEQ ID No. 52; SEQ ID No. 53; SEQ ID No. 54; SEQ ID No. 55; SEQ ID No. 56; SEQ ID No. 57; SEQ ID No. 58; SEQ ID No. 59; SEQ ID No. 60; SEQ ID No. 61; SEQ ID No. 62; SEQ ID No. 63; SEQ ID No. 64; SEQ ID No. 65; SEQ ID No. 66; SEQ ID No. 67; SEQ ID No. 68; and SEQ ID No. 69.

10. The method of claim l, wherein said HS 19 PCR primers recognize HS19 Penner serotype; HS 63 PCR primers recognize HS63 Penner serotype; HS33 PCR primers recognize HS33 and HS35 Penner serotypes; HS57 PCR primers recognize HS57 Penner serotype; HS12 PCR primers recognize HS12 Penner serotype; HS27 PCR primers recognize HS27 Penner serotype; HS21 PCR primers recognize HS21 Penner serotype; HS31 PCR primers recognize HS31 Penner serotype; HS62 PCR primers recognize HS62 Penner serotype; HS62 PCR primers recognize HS62Penner serotype; HS45 PCR primers recognize HS45 Penner serotype; HS29 PCR primers recognize HS29 Penner serotype; HS22 PCR primers recognize HS22 Penner serotype; HS9 PCR primers recognize HS9 Penner serotype; HS37 PCR primers recognize HS37 Penner serotype; HS18 PCR primers recognize HS18 Penner serotype; HS58 PCR primers recognize HS58 Penner serotype; HS52 PCR primers recognize HS52Penner serotype; HS60 PCR primers recognize HS60 Penner serotype; HS55 PCR primers recognize HS55 Penner serotype; HS32 PCR primers recognize HS Penner serotype; HS11 PCR primers recognize HS11 Penner serotype; HS40 PCR primers recognize HS40 Penner serotype; and HS38 PCR primers recognize HS38 Penner serotype.

11. The method of claim 2 , wherein the amplification of products are analyzed by agarose gel electrophoresis.

12. The method of claim 4 , wherein said PCR primer pairs are grouped into an alpha mix; a beta mix; a gamma mix and a delta mix, wherein each of said mixes comprise PCR primer pairs so that each PCR product within a mix differs by at least 20bp.

Assignees

• Poly Federic
• Guerry Patricia
• Parker Craig
• Us Navy

Inventors

• Poly Federic
• Guerry Patricia
• Parker Craig

Classifications

• C12Q1/689 Primary

  for bacteria · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title