CN115341016A - Double-end hybridization nucleic acid detection gene chip - Google Patents

Abstract

The invention discloses a gene chip for detecting double-end hybrid nucleic acid, which consists of a nucleic acid detection probe fixed on a glass slide, a polymerase chain reaction forward primer, a reverse primer and a terminal hybrid probe with a fluorescent label, wherein the 5 'end of the polymerase chain reaction forward primer contains an oligonucleotide sequence which is complementary with the terminal hybrid probe with the fluorescent label, and the 5' end of the reverse primer contains an oligonucleotide sequence which is the same as the terminal hybrid probe with the fluorescent label. The gene chip for detecting double-end hybrid nucleic acid described by the invention can effectively reduce the cost of the traditional chip for synthesizing the polymerase chain reaction primer with the fluorescent label and can improve the detection efficiency after nucleic acid hybridization.

Description

Gene chip for detecting double-end hybrid nucleic acid

Technical Field

The invention relates to the field of molecular biology, in particular to a nucleic acid detection technology based on DNA hybridization, and especially relates to a gene chip technology for detecting double-end hybridization nucleic acid.

Background

Nucleic acid hybridization is a nucleic acid detection technique commonly used in molecular biology, and is widely used for detection of microbial pathogens, antibiotic resistance genes, functional genes related to specific traits, and the like. The gene chip designed based on the nucleic acid hybridization technology can simultaneously analyze thousands of nucleic acid sequences, can simultaneously realize the rapid detection of various pathogenic bacteria and antibiotic resistance genes in a short time, and has wide application prospects in the fields of clinical medicine, animal husbandry and veterinary, fishery, agriculture and the like.

However, the conventional gene chip is labeled with fluorescence at one end of the primer, and then hybridized with the oligonucleotide probe on the chip, and then the result is detected by using a fluorescence microscope or a gene chip scanner. The fluorescent labeling of one end of the primer not only reduces the amplification efficiency of the primer, but also increases the synthesis cost of the fluorescent-labeled primer because a plurality of fluorescent-labeled primers need to be designed when a plurality of nucleic acid sequences which need to be amplified by different primers are detected simultaneously. In addition, only one of the forward and reverse primers is usually fluorescently labeled, and after the amplification product is hybridized on the gene chip, each PCR product only carries one fluorescent molecule, but the detection is performed by a general detection instrument when the emitted light of the fluorescent molecule reaches a certain intensity, thereby reducing the detection efficiency of the technology. The above defects result in high cost of gene chip detection technology, low detection efficiency and high false positive result. How to overcome the defects of the traditional gene chip technology is the core problem of developing a new nucleic acid detection gene chip technology and is also the premise of improving the wide application of the gene chip technology to nucleic acid detection.

Disclosure of Invention

The invention provides a double-end hybridization nucleic acid detection gene chip, which not only can reduce the synthesis cost of a fluorescence labeling probe, but also can improve the detection efficiency by hybridizing fluorescence labels at two ends of a PCR product through a double-hybridization technology. The double-end hybrid nucleic acid detection gene chip consists of a nucleic acid detection probe fixed on a glass slide, a polymerase chain reaction forward primer, a polymerase chain reaction reverse primer and a terminal hybrid probe with a fluorescent label. FIG. 1 depicts a schematic diagram of a double-ended hybrid nucleic acid detecting gene chip disclosed in the present patent, wherein the number of hybridization probe clusters is drawn for the purpose of more convenient illustration of the technology of the present patent and is not intended to limit the technical scope of the double-ended hybrid nucleic acid detecting gene chip disclosed in the present patent; the sequence of the primer described in FIG. 1, both ends of which are complementary to the probe, is also introduced for the convenience of describing the present patent technology, and is not a technical limitation of the double-end hybridization nucleic acid detection gene chip described in the present patent, although the sequence can be used in the double-end hybridization nucleic acid detection gene chip described in the present patent, as shown in example 1.

The nucleic acid detecting probe immobilized on the slide glass is an oligonucleotide probe designed based on the nucleic acid sequence to be detected and complementary to the nucleic acid sequence to be detected, and is modified at the 3' end with NH 2C 6 to increase the immobilization of the probe on the slide glass.

The forward and reverse primers for polymerase chain reaction are designed according to the nucleic acid sequence to be detected, and can be designed into specific primers or universal primers. The forward primer contains at the 5 'end an oligonucleotide sequence complementary to the sequence of the terminal hybridization probe with the fluorescent label, and the reverse primer contains at the 5' end an oligonucleotide sequence identical to the sequence of the terminal hybridization probe with the fluorescent label (FIG. 1).

The terminal hybridization probe with the fluorescent label is an oligonucleotide sequence with the fluorescent label, the sequence is complementary with the oligonucleotide sequence carried by the 5 'end of the forward primer and is the same as the oligonucleotide sequence carried by the 5' end of the reverse primer; thus, both ends of the nucleic acid sequence amplified by the pair of primers carry nucleotide sequences complementary to the oligonucleotide sequences carried by the fluorescently labeled terminal hybridization probes, which can hybridize with the fluorescently labeled terminal hybridization probes during the hybridization process, thereby hybridizing the fluorescent labels to both ends of the polymerase chain reaction product (FIG. 1).

In addition, two positive control probes are designed on the glass slide, wherein one of the two positive control probes is an oligonucleotide probe which is complementary to an oligonucleotide sequence carried on a terminal hybridization probe with a fluorescent label and is used for representing the hybridization effect of the gene chip; the other is a polymerase chain reaction positive amplification control; is used to characterize the hybridization effect of the amplification product on the gene chip. Each row of the chip contains at least one positive control probe, and every two rows of the chip must contain two positive control probes simultaneously; the number of positive control probes in each row is determined by the number of probes contained in each row, typically one positive control probe for every fifty to one hundred detection probes, and at least one positive control probe if less than fifty detection probes are contained in each row.

Drawings

FIG. 1 is a schematic diagram of a gene chip for detecting double-stranded nucleic acid according to the present invention.

FIG. 2 is a graph showing the results obtained in patent example 1 of the present invention.

Detailed Description

The following examples are further illustrative of the present patent and are not intended to limit the technical parameters of the present patent.

Example 1

The method described by the invention is adopted to design a method for detecting Escherichia coli: (A), (B) and (C)Escherichia coli) A universal chip. Then, the method described by the invention is adopted to design a forward bacterial universal primer Bact-F and a reverse bacterial universal primer Bact-R, and a tetracycline antibiotic resistance gene specific forward primer TetF and a reverse primer TetR. As shown in FIG. 2, the A1 site is an oligonucleotide probe complementary to an oligonucleotide sequence carried on a terminal hybridization probe having a fluorescent label. The A2 and B1 sites are the result of hybridization of the fragment obtained by PCR amplification using DNA extracted from E.coli culture as a template, and can be considered as the second positive control described in the present invention. And B2 site is negative control of polymerase chain reaction amplification by replacing a DNA template with sterile water, B6 site is probe-free, and only DNA extracted from the Escherichia coli culture is used as a template to perform PCR amplification to obtain a fragment and a hybridization probe with a fluorescent molecular marker to perform hybridization to obtain a result. The B5 site is the hybridization result of the positive amplification product of the tetracycline antibiotic resistance gene, and can also be considered as a second positive control described in the present invention. The sites A3, A4 and A5 are results obtained by hybridizing DNAs extracted from three excrement samples by adopting Bact-F and Bact-R primer amplification products; the A6, B3 and B4 sites are the results obtained by hybridizing DNA extracted from the three fecal samples by using primer amplification products of TetF and TetR. The results show that the gene chip for detecting double-end hybrid nucleic acid can effectively detect the contents of Escherichia coli and tetracycline antibiotic resistance genes in three fecal samples.

Claims (3)

1. A gene chip for detecting double-end hybrid nucleic acid is characterized in that: consists of nucleic acid detecting probe fixed on glass slide, forward and reverse PCR primers and end hybridizing probe with fluorescent label.

2. The polymerase chain reaction forward and reverse primers of claim 1 wherein: the forward primer and the reverse primer can amplify a nucleic acid sequence to be detected, the 5 'end of the forward primer contains an oligonucleotide sequence which is complementary to a terminal hybridization probe sequence with a fluorescent label, and the 5' end of the reverse primer contains an oligonucleotide sequence which is the same as the terminal hybridization probe sequence with the fluorescent label.

3. The fluorescently labeled end-hybridization probe described in claim 1, wherein: a fluorescent substance-labeled oligonucleotide sequence complementary to the specific oligonucleotide sequence contained at the 5' -end of the forward primer of the polymerase chain reaction.

Images