Multiplex amplification reaction method for determination of Campylobacter jejuni Penner/capsule type
Inventors
Poly, Frederic • Guerry, Patricia • Mason, Carl • Serichantalergs, Oralak
Assignees
Publication Number
US-8530166-B2
Publication Date
2013-09-10
Expiration Date
2031-02-22
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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 the inventive primers in a multiplex PCR assay.
Core Innovation
The invention provides a molecular method for determining Campylobacter jejuni capsule/Penner types by using multiplex DNA primers in a polymerase chain reaction (PCR) assay. This approach allows identification of C. jejuni types based on amplification of specific DNA sequences within the polysaccharide capsule (CPS) loci, rather than through traditional serological Penner typing. The method includes use of primer sets targeting regions of the capsule locus that are specific to Penner serotypes or complexes, enabling differentiation of at least 14 capsule/Penner types corresponding to 17 serotypes.
The problem addressed by this invention stems from limitations of conventional Penner serotyping, which is technically difficult, expensive, and requires production of polyclonal antisera and expression of capsule antigen, which is subject to phase variation. Additionally, Penner serotyping groups many serotypes into complexes with overlapping reactions. Prior molecular methods based on lipooligosaccharide (LOS) typing have shown limited correlation to Penner types and require amplification of large DNA fragments that are difficult to produce reliably.
The inventive method overcomes these issues by providing a simpler, more accessible, and standardized PCR-based typing scheme. It does not require capsule expression, thereby avoiding problems of phase variation and capsule shutdown. The multiplex PCR amplifies fragments smaller than 1 kb, suitable for routine molecular biology laboratories worldwide. The invention also involves designing primer pairs specific for variable regions of the C. jejuni capsule locus, particularly between conserved genes kpsC and kpsF, to capture unique sequences corresponding to Penner serotypes or complexes.
Claims Coverage
The patent includes several claims focusing on a PCR-based method for identifying Campylobacter jejuni strains by targeting specific genes in the capsule polysaccharide locus. The independent claim outlines the overall method, while dependent claims specify serotype targets, primer sequences, multiplex arrangements, and sample types.
Identification of Campylobacter jejuni by PCR targeting polysaccharide capsule loci
A method of identifying C. jejuni strains in a sample by PCR amplification using primer pairs that target one or more regions of the O-methyl phosphoramidate synthesis region, heptose synthesis region, and hyper-variable region of the polysaccharide capsule locus, followed by analysis of the amplification products.
Use of serotype-specific primer pairs for precise identification
Utilization of PCR primers designed to recognize specific penner serotypes or complexes (e.g., HS1, HS2, HS3, HS4 complexes, HS6, HS8/17, HS10, HS15/31, HS23/36, HS41, HS42, HS44, HS53, HS1 complex, and strain 8486), enabling discrimination among Campylobacter jejuni capsule types.
Multiplex PCR amplification with primer groups arranged by product size
The PCR primer pairs are grouped into at least two mixes (alpha and beta), each comprising multiple primer pairs designed for multiplex PCR to enable simultaneous amplification of different CPS loci regions. Primer groups are arranged to produce amplification products with distinguishable sizes for accurate serotype identification.
Analysis of amplification products by size determination
The method includes analyzing the PCR amplification products by determining their size, preferably using agarose gel electrophoresis, to identify the specific serotype based on unique product lengths.
Application to clinical and various sample types
The method applies PCR amplification to DNA samples obtained from various matrices, including bacterial cultures, blood, tissue, and fecal material, accommodating clinical sample types suspected of containing C. jejuni DNA.
The claims collectively cover a multiplex PCR-based method for identification of Campylobacter jejuni capsule/Penner types by targeting specific regions of the polysaccharide capsule locus, using serotype-specific primer pairs arranged in multiplex mixes, analyzing PCR product sizes for typing, and applying the method to diverse sample types including clinical specimens.
Stated Advantages
Simplifies the technically difficult and expensive process of Penner serotyping by replacing it with a molecular PCR-based approach.
Enables widespread accessibility since amplification methods are broadly available to research and reference laboratories.
Avoids dependence on capsule expression, thus unaffected by phase variation or capsule shutdown, unlike serological methods.
Multiplex PCR reduces the number of reactions needed per sample, increasing efficiency.
Utilizes amplification of DNA fragments under 1 kb, facilitating routine performance in molecular biology labs globally.
Standardized reagents (primers) allow for reproducible and cost-effective typing.
Documented Applications
Typing and identification of Campylobacter jejuni capsule/Penner types in clinical, bacterial culture, blood, tissue, and fecal samples.
Use as an alternative to serological Penner typing for epidemiological and diagnostic studies of Campylobacter jejuni infections.
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