Increased specificity of analyte detection by measurement of bound and unbound labels
An analyte and labeling technology, applied in the direction of analyzing materials, material excitation analysis, measuring devices, etc., can solve problems such as large prediction errors
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[0088] Sample preparation or pretreatment is determined by the assay method. For example, where detection using SE(R)RS is involved, the sample may be in any suitable form, such as a solid, solution or suspension, or a gas, suitably prepared to enable recording of its SE(R)RS spectrum. The test sample can be at any suitable pH.
[0089] According to a particular embodiment of the invention, the analyte is a nucleic acid, such as a genomic DNA sequence or a nucleic acid from a pathogenic microorganism. Various methods are available for isolating nucleic acid from a sample. Exemplary nucleic acid isolation techniques include (1) organic extraction followed by ethanol precipitation, e.g., using phenol / chloroform organic reagents (e.g., Ausbel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, New York (1995, including 2003 6 Supplement of August), preferably using an automated DNA extractor, such as the Model 341 DNAExtractor available from Applied Biosy...
Embodiment 1
[0105] Example 1. Introduction of internal standard in HIV detection
[0106] In this example, HIV detection is based on the presence of the gag gene (analyte) in the sample (as described by Isola et al., 1998, Anal. Chem. 70: 1352-1356).
[0107] Cresyl violet (CFV) was used as a marker, which was introduced into the analyte during PCR amplification using gag-specific oligonucleotide primers that had been labeled with CFV (as described by Isola et al. above) .
[0108] According to the present invention, a predetermined amount of CFV-labeled gag-specific oligonucleotides is used in the PCR reaction.
[0109] Design a capture probe, which is a nucleotide sequence specific to the gag gene in the amplified PCR product sequence, but different from the sequence of the PCR primer, and which has a linker capable of binding to a solid support such as a derivatized polystyrene plate (eg six carbon 5'-amino linkers). Then, the capture probes are spotted onto the support.
[0110]...
Embodiment 2
[0113] Example 2. Introducing an internal standard in the detection of chlamydia
[0114] In this example, the detection of the pathogen Chlamydia trachomatis is based on the presence of the ompl gene sequence (analyte) in the sample.
[0115] The omp1 gene in the samples was amplified by PCR using a forward primer and a reverse primer labeled with 5'-ends with biotin in the first well.
[0116] The 17-base omp1-specific DNA oligonucleotide was dyed at the 5'-end with a substituted fluorescein dye 2,5,1',3',7',9'-hexachloro-5-carboxyfluorescein (can Commercially available, referred to as "HEX") mark. The resulting HEX-labeled ompl-specific oligonucleotide ("HEX" probe) has a sequence in the sequence of the amplified ompl PCR product but differs from (nested) the sequence of the PCR primers, and the label is compatible with the organism introduced into it. The strands of the primed primers are complementary.
[0117] A predetermined amount of HEX probe was used to hybridiz...
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