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Oligonucleotides and methods for detecting Borrelia burgdorferi

a technology of oligonucleotides and borrelia, which is applied in the field of oligonucleotides and methods for detecting borrelia burgdorferi, can solve the problems of low detection efficiency, low detection efficiency, and low detection efficiency of borrelia burgdorferi, and achieve the effect of positive control

Inactive Publication Date: 2007-07-26
QUEST DIAGNOSTICS INVESTMENTS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011] In various embodiments of the present invention, oligonucleotide primers and probes are used in the methods described herein to provide the Borrelia burgdorferi assay. Thus, in certain embodiments, the invention relates to primer sequences that can be used to amplify FlaA gene in the Borrelia burgdorferi gene sequence present in a sample. The FlaA gene in the Borrelia burgdorferi gene sequence encodes the flagellin protein. In addition, primers can also be used to amplify one or more control nucleic

Problems solved by technology

Titers of specific antispirochetal antibodies (first IgM, then IgG) can be determined by ELISA or by indirect immunofluorescence, but are not useful before the patient has made antibodies.
In addition, false-positive results can be high.
Moreover, serological testing does not recognize the presence of the spirochete itself, but rather the host's immunological response to the organism following a recent or past infection.
Culture of Borrelia burgdorferi from blood and other body tissues is possible, but the recovery rate is low, and it may require many weeks before growth of the organism is evident.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 2

MagNA Pure Extraction of Borrelia burgdorferi DNA

[0067] Pre-extraction processing of specimens: Specimens should be prepared under a biohood. All instruments including pipettors, pipettes, bulbs, etc. should be used only for sample preparation and should be maintained in the hood or clean area at all times.

[0068] For pre-extraction processing of blood, CSF, SF, and urine samples and controls, a vial of HiPSP, LoPSP, NSP and a sample specimen were thawed and vortexed for 5 seconds, and then 200 μl of each was placed into their corresponding position in a MagNA Pure sample cartridge.

[0069] For ticks, the organisms were processed as follows: pipette 50 μl of PBS into each sample tube, place tick in appropriately labeled tubes, cut tick into pieces with scissors, and add additional 300 μl PBS; add 20 μl 10% SDS and 15 μl Proteinase K solution (15 μg / μl) to each sample tube and each control tube, vortexing for 5 seconds. The tubes were incubated at 65° C. for 30 min in a heat block, c...

example 3

Preparation for Borrelia burgdorferi Real-time PCR and Fluorogenic Probe Hybridization

[0072] A master mixture of reagents for performing PCR, and further hybridization with the fluorogenic probe was prepared as shown in Table 1. The mixture was dispensed in 1.0 ml aliquots and stored at −20° C.. The fluorescein dyes useful in the instant invention include 6-carboxyfluorescein (6-FAM), 5′-dichloro-6-carboxyrhodamine (JOE), and 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC). Another preferred class of labels include quencher moieties. The emission spectra of a quencher moiety overlaps with a proximal intramolecular or intermolecular fluorescent dye such that the fluorescence of the fluorescent dye is substantially diminished, or quenched, by fluorescence resonance energy transfer (FRET). Oligonucleotides which are intramolecularly labeled with both fluorescent dye and quencher moieties are useful in nucleic acid hybridization assays, e.g. the “Taqman” exonuclease-cleavag...

example 4

Data Analysis and Reporting

[0080] An assay flowchart for Borrelia burgdorferi DNA quantitative PCR is shown in FIG. 1. An average threshold cycle (Ct) value will be assigned for each well. The Ct value indicates the cycle at which exponential growth of a PCR product is occurring. If the Ct value is 43, it means that no amplification of the target was achieved. If a well has a Ct value under 43, the target has been amplified, and the PCR is thus considered to be positive.

[0081] The Ct value for the HiPSP control will generally be approximately 25.00 and the LoPSP control. should have a Ct value of approximately 32.00. The NSP should have a Ct value of 43.00.

[0082] All assay controls should be examined prior to interpretation of clinical results. If the controls are not valid, the clinical samples should not be interpreted. All positive specimen controls must be positive (i.e. have Ct value below 43) for the assay to be valid.

[0083] A positive internal control (IPC) was included w...

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PUM

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Abstract

The present invention provides methods and compositions for determining the presence and / or amount of Borrelia burgdorferi nucleic acids in a test sample related to Lyme disease. In particular, substantially purified oligonucleotide primers and probes are described that can be used for qualitatively and quantitatively detecting Borrelia burgdorferi nucleic acid in a test sample by amplification methods. The present invention also provides primers and probes for generating and detecting control nucleic acid sequences that provide a convenient method for assessing internal quality control of the Borrelia burgdorferi assay.

Description

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS [0001] This application is a Division of U.S. application Ser. No. 10 / 011,340, filed Dec. 04, 2001, incorporated herein by reference in its entirety.FIELD OF THE INVENTION [0002] The present invention relates generally to compositions and methods for detecting nucleic acids for the organism Borrelia burgdorferi in a test sample. BACKGROUND OF THE INVENTION [0003] The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention. [0004] Lyme disease, also known as Lyme borreliosis, is a tick-transmitted, spirochetal, inflammatory disorder causing a rash (erythema [chronicum] migrans) that may be followed weeks to months later by neurologic, cardiac, or joint abnormalities. Lyme disease was recognized in 1975 because of close clustering of cases in Lyme, Connecticut. It has since been reported in m...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34C07H21/04C12Q1/689
CPCC12Q1/689C12Q2600/166Y02A50/30
Inventor EXNER, MAURICEHAMDAN, HASNAHLEWINSKI, MICHAEL
Owner QUEST DIAGNOSTICS INVESTMENTS INC
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