Multiplex amplification reaction method for determination of Campylobacter jejuni Penner/capsule type
Inventors
Poly, Federic • Guerry, Patricia • Parker, Craig
Assignees
US Department of Agriculture USDA • US Department of Navy
Publication Number
US-9328389-B2
Publication Date
2016-05-03
Expiration Date
2033-08-09
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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 primers in a multiplex PCR assay.
Core Innovation
The invention provides a molecular method and associated reagents to determine Campylobacter jejuni capsule/Penner types using a multiplex polymerase chain reaction (PCR) assay. This method identifies Campylobacter jejuni types through amplification of specific DNA sequences in the capsule loci, replacing serological Penner typing by using primers that amplify type-specific sequences.
The problem addressed is the technical difficulty and expense of Penner serotyping, which requires production of type antisera and is only routinely performed in a few reference laboratories. Penner serotyping is complex, laborious, and limited by phase variation in capsule expression, making strain differentiation challenging. Previous molecular methods, such as restriction fragment length polymorphism (RFLP) of lipooligosaccharide loci, have not replaced Penner serotyping due to their technical limitations and lower correlation with Penner types.
The inventive molecular approach uses multiplex PCR primers targeting the Campylobacter jejuni capsule loci, enabling identification of 23 serotypes without the need for capsule expression. The molecular method simplifies strain identification by amplifying smaller DNA fragments (<1 kb) compared to previous large fragment PCR methods, is more easily standardized, less expensive, and is not affected by phase variation or slip-strand mutations affecting capsule expression.
Claims Coverage
The patent discloses one independent claim encompassing a method for identifying Campylobacter jejuni strains using PCR amplification of specific genes within the polysaccharide capsule loci. The following inventive features are extracted from this claim.
Identification of Campylobacter jejuni strains by PCR targeting capsule loci
A method of identifying C. jejuni strains in a sample through PCR amplification products derived from genes within the polysaccharide capsule loci, specifically targeting regions including the O-methyl phosphoramidate synthesis region, heptose synthesis, and hyper-variable region of the capsule loci.
Analysis of amplification products by size determination
The amplification products obtained by PCR are analyzed by determining their size, allowing discrimination of C. jejuni Penner serotypes based on product length.
Use of specific PCR primer pairs with defined sequences
Employing PCR primer pairs with sequences selected from a defined set of SEQ ID Nos. 1 to 46, designed to specifically amplify target regions within the capsule loci for strain identification.
Multiplex amplification approach
The method includes the use of multiplex PCR reactions, allowing simultaneous amplification of multiple target sequences in one reaction to distinguish different serotypes efficiently.
Grouping primers into distinct mixes for discrimination
Primers are grouped into alpha, beta, gamma, and delta mixes to enable separation of the amplification products by size with at least a 20 bp difference, aiding in clear discrimination of products.
Use of the method on various sample types
Application of the method to clinical and environmental samples, including bacterial cultures, blood, tissue, and fecal material suspected of containing Campylobacter jejuni DNA.
Primer design parameters
Primers contain about 18-30 nucleotides, have a G/C content of 20-50%, and melting temperatures between about 57°C and 63°C to ensure efficient and specific amplification.
Correlation of primer sets with specific Penner serotypes
Each PCR primer set is matched to recognize specific Penner serotypes or complexes, including HS19, HS63, HS33/35, HS57, HS12, HS27, HS21, HS31, HS62, HS45, HS29, HS22, HS9, HS37, HS18, HS58, HS52, HS60, HS55, HS32, HS11, HS40, and HS38.
The independent claim broadly covers a PCR-based method using specific primer combinations targeting capsule loci genes for multiplex identification of multiple Campylobacter jejuni Penner serotypes by analyzing amplification product sizes, applicable to various sample types.
Stated Advantages
The method is simpler, more easily standardized, and less expensive than traditional Penner serotyping.
It does not require capsule expression, so it is unaffected by phase variation or capsule shutdown.
Multiplex PCR reduces the number of reactions needed per sample, improving efficiency.
The method amplifies shorter DNA fragments (<1 kb), making PCR easier and more reliable than previous methods amplifying large fragments.
The method enables identification of 23 Penner serotypes, broadening typing capability.
Amplification reactions are more accessible to research and reference laboratories than serotyping antisera.
Documented Applications
Identification and typing of Campylobacter jejuni strains via their capsule/Penner serotypes in clinical samples.
Typing of Campylobacter jejuni strains from bacterial cultures, blood, tissue, and fecal materials.
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