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An oligonucleotide and chip technology, applied in the field of biochips and diagnostic reagents, achieves the effect of high specificity and wide application prospects
Inactive Publication Date: 2009-11-25
SHANDONG MEDICAL BIO TECH RES CENT
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However, in the retrieved oligonucleotide chips, there is no random primer method for simultaneous labeling of biotin-11-dUTP on the target sequences of various pathogens that cause central nervous system diseases
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Embodiment 1
[0057] Example 1 biotin-avidin-cy3 system random primer PCR amplification labeled oligonucleotide chip
[0058] 1. Design and synthesis of probes
[0059] Using the probe design software ArrayDesigner4.2, according to the principle of probe design, the G+C content is 50%-70%; the Tm value is at (78±5)°C; the number of consecutively repeated bases is ≤6; the internal complementary base of the probe molecule bases ≤ 6; the similarity between each probe and the non-target gene must be <70%, the number of consecutive homologous bases of the probe ≤ 18, the hairpin structure, dimer and mismatch as little as possible, melting Probes with temperature (TM value) values as close as possible; two probes (CMV-1, CMV-2, HSVI-1, HSVI-2, RV1, RV2, EBV1, EBV2, TB1 and TB2), and design a positive control probe (PC) and a negative control probe (NC) at the same time; the probe sequences are as follows:
[0090] The optimization of embodiment 2 chip preparation conditions
[0091] 1.1 The optimization of the hydration temperature selects the CMV standard strain DNA amplification marker, uses the aldehydized glass slide as the substrate, and selects the energy intensity of ultraviolet crosslinking at 37°C, 55°C and 65°C for the hydration temperature. The temperature with the best hybridization effect was selected as the hydration temperature.
[0092] After 3 repeated experiments, it was shown that the hybridization effect was best when the hydration temperature was 37°C.
[0093] 1.2 Optimization of UV cross-linking DNA amplification markers of CMV standard strains were selected, aldehydized glass slides were used as substrates, and the energy of UV cross-linking was selected as 600mJ, 1200mJ and 3600mJ respectively, and the energy result with the best hybridization effect was selected.
[0094] After 3 repeated experiments, it was shown that the energy of UV crosslinking was ...
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Abstract
The invention discloses an oligonucleotide chip labeling a random primer PCR amplification based on a biotin-avidin-cy3 reaction system, which is made by a substrate, and CMV, HSVI, RV, EBV or TB pathogen probes, a negative control, a positive control and a blank control that are coated on the substrate in an array way, wherein the substrate is a surface aldehyde glass slide; the pathogen probes are 10 of CMV-1, CMV-2, HSVI-1, HSVI-2, RV1, RV2, EBV1, EBV2, TB1 and TB2 probes with 70mer; the positive control is a synthesized marker probe; and the blank control is double distilled water. The invention performs the biotin-11-dUTP amplified labeling to various pathogen target sequences by using the random primer and detects the pathogen by adopting a method of increasing the labeling efficiency through using a biotin-avidin-cy3 signal amplification system, has high detection sensitivity and obviously shortens the detection cost and the detection time. The technical system can be suitable to the fields of clinical detection, health supervision, customs quarantine control, scientific research, and the like.
Description
technical field [0001] The present invention relates to an oligonucleotide chip and its application, in particular to a biotin-avidin-cy3 reaction system-based random primer PCR amplification-marked oligonucleotide chip and its application in the detection of central nervous system pathogens , which belongs to the technical field of biochips and diagnostic reagents. Background technique [0002] Central nervous system infectious disease is a disease caused by a variety of pathogens that seriously threatens human life and health. This type of disease is acute, difficult to diagnose, and has a high fatality rate. This requires rapid and accurate detection of pathogens in cerebrospinal fluid. Identification, in order to take effective targeted treatment measures early. Due to different pathogenic infections, the scope and degree of lesions vary greatly, and the clinical manifestations are very complicated, which brings difficulties to diagnosis. The main basis for diagnosis is...
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