Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1992-10-09
1999-06-15
Marschel, Ardin H.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 435810, 436501, 536 231, 536 241, 536 243, 536 2431, 536 2432, 536 2433, 935 77, 935 78, C12Q 168
Patent
active
059121173
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The invention relates generally to methods and reagents for the detection of Lyme disease, a multisystem bacterial infection transmitted by Ixodid ticks. More particularly, the methods employ polymerase chain reaction to amplify genomic nucleic acid sequences specific to the etiologic agent of Lyme disease, Borrelia burgdorferi.
BACKGROUND OF THE INVENTION
Lyme disease is a complex multisystem disorder caused by the tick-borne spirochete Borrelia buradorferi. This disease has three clinical stages that can overlap or occur alone: stage one--early disease, including a characteristic expanding skin lesion (erythema chronicum migrans) and constitutional flu-like symptoms; stage two--cardiac and neurological disease; and stage three--arthritis and chronic neurological syndromes.
Presently, the incidence of reported Lyme disease is increasing, which is probably due to improved awareness and recognition of the disease, as well as to an actual increase in incidence and geographic spread. B. burgdorferi can be isolated from blood or skin biopsies taken from acutely ill patients, but the yield is low and the procedures are difficult. Serologic testing for antibodies using an enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence assay (IFA) is the standard method used to confirm a clinical diagnosis, but current tests are poorly standardized, and false-negative or false-positive results can occur (Barbour, (1989) Ann Intern Med 110:501). In addition to misdiagnosis caused by lack of standardization of serologic testing, cross-reactivity with Treponema and with other Borrelia may occur. Patients with stage one or two disease may be seronegative because it may take as long as three to six months after exposure for antibodies to become detectable with currently available tests. Patients who develop later stages of the illness may occasionally be seronegative if they were treated acutely with antibiotics (Dattwyler et al., (1988) N Engl J Med 319:1441). Previously untreated patients with a late stage of the disease are, apparently, almost always seropositive.
The use of specific polynucleotide sequences as probes for the recognition of infectious agents is becoming a valuable alternative to problematic immunological identification assays. For example, PCT publication W084/02721, published Jul. 19, 1984 describes the use of nucleic acid probes complementary to targeted nucleic acid sequences composed of ribosomal RNA, transfer RNA, or other RNA in hybridization procedures to detect the target nucleic acid sequence. While the assay may provide greater sensitivity and specificity than known DNA hybridization assays, hybridization procedures which require the use of a complementary probe are generally dependent upon the cultivation of a test organism and are, therefore, unsuitable for rapid diagnosis.
Polymerase chain reaction (PCR) is a powerful technique that can be used for the detection of small numbers of pathogens whose in vitro cultivation is difficult or lengthy, or as a substitute for other methods which require the presence of living specimens for detection. In its simplest form, PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA. A repetitive series of cycles involving template denaturation, primer annealing, and the extension of the annealed primers by DNA polymerase results in the exponential accumulation of a specific fragment whose termini are defined by the 5' ends of the primers. PCR reportedly is capable of producing a selective enrichment of a specific DNA sequence by a factor of 10.sup.9. The PCR method is described in Saiki et al., (1985) Science 230:1350 and is the subject of U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188. This method has been used to detect the presence of the aberrant sequence in the beta-globin gene which is related to sickle cell anemia (Saiki et al., (1985) supra) and the human im
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Dodge Deborah E.
White Thomas J.
Johnston George W.
Marschel Ardin H.
Petry Doug
Roche Molecular Systems Inc.
Tramaloni Dennis P.
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