Compositions, kits and methods for detection of campylobacter nucleic acid

#This invention relates to detection of the presence of Campylobacter jejuni (C. jejuni), Campylobacter lari (C. lari), or Campylobacter coli (C. coli) bacteria in a sample by using molecular biological methods, and specifically relates to detection of C. jejuni, C. lari, or C. coli in a sample by amplifying nucleic acids from C. jejuni, C. lari, or C. coli and detecting the amplified nucleic acid sequences.

#Campylobacter contamination is one of the major causes of food-borne illness, and can lead to Campylobacteriosis. Campylobacter are also a major cause of diarrhoeal illness in humans and are generally regarded as the most common bacterial cause of gastroenteritis worldwide. In the United States, Campylobacter are estimated to affect two million people a year. Common routes of transmission are ingestion of contaminated food, water or milk, eating raw meat, person-to-person sexual contact, and fecal-oral. The infectious dose for Campylobacter may be as low as 400-500 bacteria as shown by human feeding studies, however, the infectious dose and the dose response are dependent upon the strains used, and the age and physical condition of the individuals. Symptoms appear 2-10 days after ingesting the bacteria and include fever, abdominal cramps, and mild to severe diarrhea, which may be bloody. Most infections are caused by C. jejuni, normally found in cattle, swine, and birds, where it is non-pathogenic, however, the illness may also be caused by C. coli (also found in cattle, swine, and birds) and C. lari (present in seabirds in particular). Some strains of C. jejuni produce a cholera-like enterotoxin, which is important in the watery diarrhea observed in infections. Most Campylobacter infections clear on their own or with the aid of antibiotics, however, long-term sequelae include reactive arthritis, Guillain-Barré syndrome and Miller Fisher Syndromes.

#Studies have shown that Campylobacter can be isolated from a wide variety of sources, including chicken, cattle, pets, goats, sheep and pond water and river water. Milk, particularly unpasteurised (raw) milk, and poultry are the most common food items associated with Campylobacter infection. Campylobacter may also be isolated from red meat, but the frequency of contamination is generally much lower than poultry. Isolation of Campylobacter from food sources is difficult because the bacteria are usually present in very low numbers since they generally do not multiply in the foods they infect. Culture methods require an enrichment broth containing antibiotics, special antibiotic-containing plates, incubation at two different temperatures, and a microaerophilic atmosphere with an elevated concentration of carbon dioxide. Isolation can take several days to a week. Identification of Campylobacter using current USDA methods for detection can take 2-3 days. There is a need for a rapid, sensitive and accurate method to detect C. jejuni, C. lari, or C. coli so that the contaminating or infectious source can be accurately detected and eliminated. There is also a need for methods that allow rapid and accurate diagnosis of C. jejuni, C. lari, or C. coli infections in humans so that infected individuals may be treated promptly to limit morbidity and prevent further infections.

#FIG. 1 shows nucleotide residues 1-450 from the 16S rRNA nucleotide sequence of Campylobacter jejuni GenBank Accession Number AF393202.1, GI:20378208 (May 1, 2002).

#FIG. 2 shows the nucleotide sequence of the 16S rRNA nucleotide sequence of Campylobacter jejuni GenBank Accession Number AF393202.1, GI:20378208 (May 1, 2002), which is also SEQ ID NO:91.

#Disclosed are compositions, kits and methods for amplifying and detecting one or more of C. jejuni, C. lari, or C. coli target nucleic acid present in a sample. These target nucleic acids are 16S rRNA and/or genes encoding 16S rRNA from one or more of C. jejuni, C. lari, or C. coli. Preferably, the samples are food samples, water samples, industrial samples, environmental samples or biological samples. The compositions, kits and methods provide oligonucleotide sequences that specifically recognize these target nucleic acids. Such oligonucleotides may be used as amplification oligonucleotides, which may include primers, promoter primers, blocked oligonucleotides, and promoter provider oligonucleotides, whose functions have been described previously (e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 5,399,491; 5,554,516; 5,824,518; and 7,374,885). Other oligonucleotides may be used as probes for detecting amplified sequences of C. jejuni, C. lari, and/or C. coli.

#The amplification step includes contacting the sample with one or more amplification oligonucleotides specific for a target sequence within the target nucleic acid. Amplification synthesizes additional copies of the target sequence or its complement by using at least one nucleic acid polymerase. Depending on the amplification method used and the configuration of the amplification oligomer, amplification synthesizes a complementary DNA or RNA sequence corresponding to the target sequence or its complement. Ordinarily skilled artisans understand the amplification process employed in the various amplification techniques, and understand that the amplification product strands are sense or antisense to the template strand and may be RNA or DNA strands. One embodiment for detecting the amplified product uses a hybridizing step that includes contacting the amplified product with at least one probe specific for a portion of the amplification product. Some other detection methods include, gel electrophoresis, dye staining, radiolabeling and mass spectrometry, to name a few.

#A detecting step may be performed after the amplification reaction is completed, or may be performed simultaneous with amplifying the target region, e.g., in real time. In one embodiment, the detection step allows homogeneous detection, e.g., detection of the hybridized probe without removal of unhybridized probe from the mixture (e.g., U.S. Pat. Nos. 5,639,604 and 5,283,174). In embodiments that detect the amplified product near or at the end of the amplification step, a linear probe may be used to provide a signal to indicate hybridization of the probe to the amplified product. In other embodiments that use real-time detection, the probe may be a linear probe, such as a dual labeled TaqMan probe detected upon 5′→3′ exonuclease degradation of the probe, or may be a hairpin probe, such as a molecular beacon, molecular torch, or hybridization switch probe labeled with a reporter moiety that is detected when the hairpin probe binds to amplified product. Various forms of such probes have been described previously (e.g., U.S. Pat. Nos. 5,118,801; 5,210,015; 5,312,728; 5,538,848; 5,925,517; 6,150,097; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and US Pub. Nos. 20060068417A1; and US Pub. No. 20060194240A1).