Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-09-06
2002-03-12
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S800000, C536S023100, C536S024310, C536S024330
Reexamination Certificate
active
06355435
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to a process for detecting and enumerating
Campylobacter jejuni
in an environmental sample. The present invention further relates to a process which can distinguish antibiotic resistant strains of
Campylobacter jejuni
from wild-type strains, in particular, antibiotic-resistant strains resistant to high levels of ciprofloxacin. Both processes relate to PCR primers which flank a target sequence unique to
Campylobacter jejuni
in combination with one or more dual-labeled oligonucleotide probes complementary to the target sequence wherein the dual-labeled probes enable detection of PCR amplification by fluorescence detection means.
(2) Description of Related Art
Campylobacter spp. is the leading cause of gasterial enteritis in the United States, affecting millions of people annually. Of the people affected,
Campylobacter jejuni
represents the etiological agent for a large proportion of the clinical cases of Campylobacter infections in human patients. Campylobacter is a gram negative microaerophilic pathogen of both humans and animals. In humans, Campylobacter infection is characterized by acute diarrheal disease, and more recently, has been associated with Guillain Barre Syndrome, a peripheral neuropathy characterized by limb weakness, and other neurological and systemic sequelae (Hughs et al., J. Infect. Dis. 176(Suppl. 2): S92-S98 (1997); Blaser, J. Infect. Dis. 176(Suppl.): S103-S105 (1997)).
Erythromycin, fluoroquinolones, and tetracyclines are classes of antibiotics that are most frequently used to treat Campylobacter infections (Blaser, J. Infect. Dis. 176(suppl. 2): S103-S105 (1997); Altkreuse et al., Emerg. Infect. Dis. 5: 28-35 (1999)). While these antibiotic treatments are effective at controlling bacterial enteritis, increasingly, antibiotic resistant strains of
Campylobacter jejuni
are being uncovered (Gaudreau et al., Antimicrob. Agents Chemother. 42: 2106-2108 (1998); Gaunt et al., J. Antimicrob. Chemother. 37: 747-757 (1996); Moore et al., Vet. Rec. 138: 306-307 (1996); Lee et al., Internatl. J. Food Microbiol. 24: 161-170 (1994); Taylor et al., Antimicrob. Agents Chemother. 32: 1107-1112 (1988); Tenover et al., Antimicrob. Agents Chemother. 27: 37-41 (1985)). The appearance of antibiotic resistant strains of
Campylobacter jejuni
is a public health concern. Therefore, much attention has been drawn to
Campylobacter jejuni
strains which are resistant to fluoroquinolones, a class of antibiotics most frequently used for treating bacterial enteritis of which ciprofloxacin is an example.
The predominant mechanism for high-level ciprofloxacin resistance (MIC equal to or greater than 16 &mgr;g/ml) in
Campylobacter jejuni
appears to be a C to T nucleotide transition in amino acid codon 86 in the quinolone resistance determining region (QRDR) of the gyrA gene. The gyrA gene encodes one subunit of DNA gyrase, which is the target for fluoroquinolone antibiotics. The amino acid codon 86 mutation results in a threonine to isoleucine amino acid substitution in the functional gyrA protein (Wang et al., Antimicrob. Agents Chemother. 37: 457-463 (1993); Charvalos et al., J. Clin. Lab. Anal. 10: 129-133 (1996); Husmann et al., J. Clin. Microbiol. 35: 2398-2400 (1997); Gibreel et al., Antimicrob. Agents Chemother. 42: 3276-3278 (1998); Ruiz et al., Microbiol. Immunol. 42: 223-226 (1998)). Ciprofloxacin susceptibility testing of Campylobacter is commonly performed using standard methods such as broth or agar dilution (Charvalos et al., ibid.; Gaunt et al., ibid.; Gaudreau et al., ibid.; Ruiz et al., ibid.) which are tedious and time consuming methods.
In order to conduct adequate epidemiological studies to determine the sources of
Campylobacter jejuni
in the agricultural environment, a sensitive diagnostic test is desirable. The appropriate test would allow execution of surveillance programs to monitor contamination of food and the environment in a timely manner. Therefore, the test most appropriate is a test of high sensitivity, specificity, and short turn-around time.
Currently, the standard method for isolation and identification of
Campylobacter jejuni
involves a pre-enrichment step in supplemental broth and incubation for between about 24 and 48 hours under suitable growth conditions. Next, there follows an isolation step by subculturing the enrichment broth onto Campylobacter selective media and incubation for an additional 24 to 72 hours. The identity of isolated colonies of Campylobacter from selective media can be confirmed using biochemical tests or polymerase chain reaction (PCR) methods. Antibiotic susceptible or resistant Campylobacter strains can be identified using particular susceptibility/resistance testing which requires an additional 24 hours for completion. Thus, to identify antibiotic-resistant strains of
Campylobacter jejuni
in an environmental sample requires an average of four and a half days to complete.
Therefore, there is a need for a test of improved sensitivity, and specificity, and further of short turn-around time that would enable detection and enumeration of
Campylobacter jejuni
in environmental samples. With the appearance of strains which are antibiotic resistant, there is a further need for a rapid and sensitive test that will detect these antibiotic-resistant strains in an environmental sample.
SUMMARY OF THE INVENTION
The present invention provides a process for detecting and enumerating
Campylobacter jejuni
in an environmental sample. The present invention further provides a process which can distinguish antibiotic resistant strains of
Campylobacter jejuni
from wild-type strains, in particular, antibiotic-resistant strains resistant to high levels of ciprofloxacin. Both processes use PCR primers which flank a target sequence unique to
Campylobacter jejuni
in combination with one or more dual-labeled oligonucleotide probes complementary to the target sequence wherein the dual-labeled probes enable detection of PCR amplification by fluorescence detection means.
Therefore, the present invention provides a process for detecting and enumerating
Campylobacter jejuni
in a sample, the process comprising: (a) providing in a PCR reaction mixture a sample suspected to contain a target nucleic acid sequence that is unique to
Campylobacter jejuni
, a first oligonucleotide PCR primer and a second oligonucleotide PCR primer which hybridize to opposite strands of the target nucleic acid sequence and flank the target nucleic acid sequence for PCR amplification of the target nucleic acid sequence, each of four deoxynucleoside triphosphates selected from the group consisting of adenosine, guanosine, thymidine, cytosine, and analogs thereof, a nucleic acid polymerase having a 5′ to 3′ exonuclease activity and lacking 3′ to 5′ exonuclease activity, and an oligonucleotide probe blocked against chain extension at its 3′ end and labeled at its 5′ with an energy transfer donor fluorophore and labeled at its 3′ end with an energy transfer acceptor fluorophore wherein the oligonucleotide probe is complementary to the target nucleic acid; (b) amplifying the target nucleic sequence in the sample under suitable PCR reaction mixture temperature conditions by a repetitive series of PCR thermal cycling steps comprising: (1) denaturing the target nucleic acid sequence into opposite strands; (2) hybridizing the first and second oligonucleotide PCR primers and the oligonucleotide probe to the denatured strands, and (3) extending the hybridized primers with the four deoxynucleoside triphosphates and the nucleic acid polymerase, and producing 5′ fluorophore and 3′ fluorophore labeled nucleotide fragments during the extension phase by the 5′ to 3′ exonuclease activity of the nucleic acid polymerase on the oligonucleotide probe annealed to the denatured strands; (c) following amplification of the target nucleic acid sequence by
Kaneene John B.
Linz John E.
Mansfield Linda S.
Newman Thomas C.
Walker Robert D.
Board of Trustees of Michigan State University
Horlick Kenneth R.
McLeod Ian C.
Spiegler Alexander H.
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