CN1546684A - All-purpose gene chip for quick detection of food security and its detection method - Google Patents

Abstract

The invention discloses an all-purpose gene chip for quick detection of food security and its detection method, which comprises arranging a molecular probe for 27 bacterial on a gene chip, carrying out qualitative and quantitative analysis simultaneously, determining gene type of polymorphism sites based on the signals of the gene type probe on the gene chip, judging the qualification of foodstuff according the detecting data and foodstuff type.

Description

A kind of gene chip of universal food safety rapid detection and detection method

The present invention relates to a kind of biotechnology, more specifically to a kind of gene chip and detection method of universal food safety rapid detection.

Nucleic acid is the genetic material of living species, and it comprises Yeast Nucleic Acid (RNA) and thymus nucleic acid (DNA).The essentially consist of nucleic acid is four kinds of Nucleotide (or base), and they are respectively by A, C, G, T representative in DNA, and they are respectively by A, C, G, U representative in RNA.Nucleotide (base) is formed chain-like structure in placed in-line mode in nucleic acid, DNA is generally duplex structure, and RNA is a single-stranded structure; The duplex structure of DNA can resolve into strand under suitable condition, and the DNA of strand also can form duplex structure with the RNA of strand.In a nucleic acid construct, a part can be that strand another part can be double-stranded.The T of base A and another chain (U) forms base pair by hydrogen bond in the nucleic acid construct of two strands, and in like manner the C of G and another chain forms base pair, and the base that can form base pair is a complementary base.As a nucleic acid chains and another nucleic acid chains complete complementary base sequence is arranged, then these two nucleic acid chains are called complementary nucleic acid chain, the complementary nucleic acid chains can form stable duplex structure, and the process that is formed the duplex molecule structure by complementary two single chain molecule structures is called molecular hybridization.Non-complete complementary nucleic acid chains (part is complementary) also can form the duplex molecule structure under given conditions.In organism and test tube, the nucleic acid chains of strand can copy the complementary nucleic acid chains under the effect of enzyme, in reproduction process, the nucleic acid chains that is replicated is called template molecule, required enzyme is a polysaccharase, and polysaccharase can be divided into archaeal dna polymerase, RNA polymerase, reversed transcriptive enzyme etc.Archaeal dna polymerase is template synthetic DNA complementary strand with DNA, and RNA polymerase is the synthetic RNA complementary strand of template with DNA, and reversed transcriptive enzyme is template synthetic DNA complementary strand with RNA.Primer is a nucleic acid fragment, and it and the complementary also hybridization of template molecule form duplex structure.The initial point of nucleic acid polymerase reaction is decided by the position of primer, and the polyreaction of DNA is from 3 ' terminal (DNA and cDNA polyreaction) of primer.The RNA polymerase reaction needed has the double-stranded structure of being discerned by RNA polymerase, is called driven element.The primer nucleic acid fragment can be made up of several or tens bases (Nucleotide), and primer can be DNA, also can be RNA, and it can be the fragment that obtains by degraded nature nucleic acid, also can be by artificial chemosynthesis.The nucleic acid fragment of synthetic can have non-natural composition unit (structure), and non-natural composition unit's (structure) is included in the structure that occurring in nature is not a nucleic acid component, also comprises not being present in natural chemical structure.By the hybridization of primer and template molecule, can the control template molecule duplicate and the amplification of template molecule.Nucleotide sequence can change during evolution with under the influence of ambient conditions, the modal base that is changed to is replaced by another base, in same species, if any different base compositions, then this a kind of phenomenon is called single base (Nucleotide) polymorphism (SNP) to the same position of same nucleotide sequence (single base position) between different individualities.In particular individual, a kind of based composition of pleomorphism site is a kind of genotype (gene is the functional region of nucleic acid) in this site, a monoploid biont can only have a genotype on a polymorphic point, in amphiploid biont, has identical genotype as two homologous genes, this individuality is a homozygote, if any different genotype, then this individuality is a heterozygote.Genotype detection is significant to genetic diagnosis, species evaluation, drug screening etc.DNA can be replicated under the effect of polysaccharase etc., promptly copy complementary strand by template strand, duplicating of DNA needs a primer, primer is generally one and contains nucleic acid chains several or tens bases, nucleic acid chains has directivity and uses 5 ' end and 3 ' end expression respectively, sequence near 5 ' end is called 5 ' end parts, the 3 ' end parts that is called near 3 ' end, primer and template are held to have specifically 3 ' and are combined into duplex structure (this process is called molecular hybridization) according to the base pairing principle before the nucleic acid replication, after this polysaccharase can be from 3 ' end beginning of primer along 5 ' → 3 ' direction synthetic with the template new chain of complementary mutually, the new two strands that forms at high temperature (＞90 ℃) is separated into independently strand, the primer molecule of remainder can be again and the template corresponding making nucleic acid molecular hybridization when temperature descends, and can under the effect of polysaccharase, duplicate the nucleic acid chains that makes new advances, as above process repeatedly, a nucleic acid fragment can be amplified out ten million same molecule fragment, and as above process is pcr process (PCR).The part that one nucleic acid samples need increase is the target fragment, carry out the PCR reaction and generally corresponding primer will be arranged at target fragment two ends, to near the single-minded primer of the sequence the polymorphic site is polymorphism primer, specific gene type to a pleomorphism site has the narrow spectrum genotype primer that is, polymorphism primer can be used in combination with the genotype primer target fragment is carried out pcr amplification, and measures genotype.With the method for chemistry can the synthetic nucleic acid fragment as primer, the base sequence of primer is complementary mutually with the target fragment sequence, can have in the primer sequence in addition with nucleic acid samples in the non-complementary sequence of sequence, this and sample are not had a sequence of complementarity and are called exogenous array, and exogenous array can be used as the part-structure of primer.Gene chip is a kind of the different IPs acid molecule to be arranged in specific position respectively, and be used for the device of making nucleic acid molecular hybridization, the nucleic acid molecule that is fixed on the gene chip is a probe molecule, with probe molecule carry out specific hybrid for target molecule, probe molecule can by in the position of chip or with mark carry out target molecule identification, mark can have various ways, as radio-labeling, fluorescent mark, nanoparticle label, enzyme labelling, pigment mark etc., be the genotype probe in order to the nucleic acid probe molecules of tool mark of difference different genotype.The polyreaction or the amplified reaction that are caused by primer are usually used in molecular biological fundamental research, genetically engineered, and genetic diagnosis and the detection relevant with molecular biology.When detecting one or more food samples, the target molecule kind number of required screening and amplification can reach hundreds of more than, as with conventional PCR method, need synthetic a large amount of primer when carrying out Molecular Detection, and respectively target molecule screened and increase with a large amount of reactions.Required man power and material, the expense of this way is all very big.If in a PCR reaction, the interaction between these primers can produce by product a plurality of combination of primers.Existing food microorganisms virus detects still along continuous traditional cultivation, separation, evaluation (identification of morphology, biochemical identification, serological identification) program, this technological operation cycle is long, every accumulative total expense height is difficult to the safety and sanitation of food are formed effective supervision.It generally is according to national food hygienic standard that existing food safety (health) detects, according to the food sanitation type such as sauce halogen meat, seasoned, cake bread, dried meat floss, dry fruit or the like, standard is numerous, but wherein most importantly must not detect pathogenic bacterium, detect pathogenic bacterium but prior art often is difficult to one one.

The gene chip and the detection method that the purpose of this invention is to provide a kind of universal food safety rapid detection, it can overcome the above-mentioned deficiency of prior art.

A kind of gene chip of universal food safety rapid detection and detection method, comprise sampling, cultivation, separation, the evaluation of food samples, it is characterized in that on a gene chip, designing the molecular probe that 27 kinds of bacterial classifications are arranged, carry out qualitative and qualitative and quantitative analysis simultaneously, according to the genotype of the signal judgement pleomorphism site of genotype probe on gene chip, contrast food type again to judge the qualified method of food according to detecting data.

Advantage of the present invention is to have shortened the cycle that food sanitation (safety) detects greatly, has enlarged sensing range, has reduced testing cost, has dwindled the detection error, has strengthened the food safety index, can form effective supervision to the safety and sanitation of food products market.

Below by embodiment the present invention is described.

Sample to certain food carries out microorganism (pathogenic bacterium, virus) check.Make the gene chip that detects food, molecular probe on the gene chip is and the exogenous array of polymorphism primer complementary nucleic acid molecule mutually, comprises Salmonellas, vibrio cholerae, Vibrio parahemolyticus, Enterohemorrhagic E.coli, listeria spp, Hemolytic streptococcus, Clostridium botulinum, bacillus cereus, Shigellae, campylobacter jejuni, yersinia entero-colitica, vibrio alginolyticus, Vibrio vulnificus, anthrax bacillus, streptococcus faecium, Brucella, foot and mouth disease virus, avian influenza virus, Avian pneumo-encephalitis virus, mad cow disease cause of disease; Coliform, excrement colibacillus group, intestinal bacteria, clostridium perfringens, streptococcus aureus, mould, saccharomycetic nucleic acid fragment (molecule).Its length is made up of 10-50 Nucleotide, each probe is fixed in the surface of chip respectively with known method, prepare a test kit, test kit comprises and is used for primer that polymorphism target fragment is increased, the segmental primer of a polymorphism target that increases comprises single-minded polymorphism primer and two single-minded genotype primers, test kit also comprises the reagent of nucleic acid purification, the enzyme of PCR reaction, test kit comprises that two or more have different marker genotypes probes respectively in addition, be labeled as the structure of the nanometer range of fluorochrome or other available chemistry or physical method identification, test kit also comprises the damping fluid that is used for molecular hybridization.When carrying out genotype detection, adopt following schedule of operation: 1. from the sample of this food, extract the DNA of microorganism, the damping fluid that the DNA of extraction is water-soluble or suitable; 2. with the PCR reaction reagent DNA nucleic acid that extracts is carried out pcr amplification, with a polymorphism primer and two single-minded genotype primers one polymorphism target fragment is increased, many group primers can use (composite PCR reaction) in same test tube, the compound degree can be two groups or two groups of above primers, product after the amplification has the polymorphism exogenous array of strand and the genotype exogenous array of strand, and the volume of reaction is generally at the 10-100 microlitre; 3. the reaction product of a plurality of PCR can merge, and carries out suitable purifying, processings (can remove unreacted primer in case of necessity) such as concentrates, and combines with the damping fluid of hybridizing usefulness then, and final volume is in 0.1-1 milliliter scope.Comprise in the hybridization solution and have different marker genotypes probes; 4. carry out gene chip hybridization and cleaning with known method and instrument, and with the signal on laser scanner or other the corresponding instrument acquisition chip; 5. according to the ratio of the intensity of position, feature and the signal of signal on chip, can measure the genotype of pleomorphism site.Can draw (27 kinds) bacterium (genus) kind whether this food has above probe design according to above detected result, promptly carried out the qualitative detection that bacterium (genus) is planted, for the pathogenic bacterium Salmonellas, vibrio cholerae, Vibrio parahemolyticus, Enterohemorrhagic E.coli, listeria spp, Hemolytic streptococcus, Clostridium botulinum, bacillus cereus, Shigellae, campylobacter jejuni, yersinia entero-colitica, vibrio alginolyticus, Vibrio vulnificus, anthrax bacillus, streptococcus faecium, Brucella, foot and mouth disease virus, avian influenza virus, Avian pneumo-encephalitis virus, the mad cow disease cause of disease is only done qualitative detection, be in this food inspection as long as there are above a kind of pathogenic bacterium, announce that promptly this food is unacceptable product.If this food does not detect above pathogenic bacterium, carry out quantitative analysis again for coliform, excrement colibacillus group, intestinal bacteria, clostridium perfringens, streptococcus aureus, mould, yeast, quantitative analysis is carried out with known technology.Draw the qualitative, quantitative assay (detection data) of microorganism in this food by above method.All inspection for food hygiene standards are input in the computer, set up one sub-category, by different level, graduate java standard library, judge the conclusion that draws this food test according to codex alimentarius again according to this food qualitative, quantitative assay (detection data).

Present method is used for the detection of food, the computer software that comprises all inspection for food hygiene standard sets, all inspection for food hygiene standards comprise international standard, national standard, industry standard and the relevant company standard of common food, with these regular sets synthetic one sub-category, by different level, graduate inspection for food hygiene standard software.The inspection for food hygiene standard software is in order to contrast food test result, the judgement of concluding property.

The check that present method is used for food is general in all food.The design of gene chip, use are that the sample of the food of being checked is responsible for, and do not consider kind, the standard of food, detect with biochip (gene chip) after the food sampling, compare the standard of certain based food after detected result is come out again and judge.

Can produce test kit according to present method, be useful on the primer that polymorphism target fragment is increased in the test kit, comprise single-minded polymorphism primer and genotype primer, and the enzyme of the reagent of nucleic acid purification, PCR reaction, damping fluid etc.

Can increase new pathogenic bacterium, pathogenic micro-organism and virus with the concrete microbial strains that detects of this method, viral species because of the passing of time and the variation of standard.

The polymeric enzyme reaction that carries out with this method is to carry out at PCR, and the quantitative PCR instrument can be observed the degree of carrying out of reaction by optical system in the PCR reaction process.

This method can be used for the RNA target molecule, carries out reverse transcription reaction and carries out polymerase chain reaction then, judges the kind and the quantity of genetic expression or mensuration RNA viruses (microorganism) with reaction result.

This method can be used for the DNA target molecule and carries out polymerase chain reaction, or the isothermal amplification reactions of being made up of reverse transcription reaction, DNA polyreaction, RNA polyreaction etc.Result by amplification judges the segmental existence of target or judges the segmental genotype of target, and the quantity of judging relevant species.

This method can detect virus and microorganism, is used for food sanitation, commodity inspection, environmental protection, medical treatment etc.

Claims (8)

1, a kind of gene chip of universal food safety rapid detection and detection method, comprise sampling, cultivation, separation, the evaluation of food samples, it is characterized in that on a gene chip, designing the molecular probe that 27 kinds of bacterial classifications are arranged, carry out qualitative and qualitative and quantitative analysis simultaneously, according to the genotype of the signal judgement pleomorphism site of genotype probe on gene chip, contrast food type again to judge the qualified method of food according to detecting data.

2, method according to claim 1 is characterized in that the biochip probe has Salmonellas, vibrio cholerae, Vibrio parahemolyticus, Enterohemorrhagic E.coli, listeria spp, Hemolytic streptococcus, Clostridium botulinum, bacillus cereus, Shigellae, campylobacter jejuni, yersinia entero-colitica, vibrio alginolyticus, Vibrio vulnificus, anthrax bacillus, streptococcus faecium, Brucella, foot and mouth disease virus, avian influenza virus, Avian pneumo-encephalitis virus, mad cow disease cause of disease; Coliform, excrement colibacillus group, intestinal bacteria, clostridium perfringens, streptococcus aureus, mould, saccharomycetic nucleic acid fragment.

3, method according to claim 1 is characterized in that qualitative and qualitative and quantitative analysis is to finish on a gene chip.

4, method according to claim 1, it is characterized in that judging the genotype of pleomorphism site according to the signal of genotype probe on biochip, 5 ' end parts of polymorphism primer and genotype primer has single-minded exogenous array respectively, 3 ' end parts of polymorphism primer and genotype primer has respectively and target sequence complementary sequence mutually, and non-naturality structure is arranged between exogenous array and the complementary sequence.

5, method according to claim 1 is characterized in that test kit is useful on the primer that polymorphism target fragment is increased, and comprises single-minded polymorphism primer and genotype primer, and enzyme, the damping fluid of the reagent of nucleic acid purification, PCR reaction.

6, method according to claim 1 is characterized in that only doing qualitative detection for pathogenic bacterium Salmonellas, vibrio cholerae, Vibrio parahemolyticus, Enterohemorrhagic E.coli, listeria spp, Hemolytic streptococcus, Clostridium botulinum, bacillus cereus, Shigellae, campylobacter jejuni, yersinia entero-colitica, vibrio alginolyticus, Vibrio vulnificus, anthrax bacillus, streptococcus faecium, Brucella, foot and mouth disease virus, avian influenza virus, Avian pneumo-encephalitis virus, mad cow disease cause of disease.

7, method according to claim 1 is characterized in that carrying out qualitative and quantitative analysis for coliform, excrement colibacillus group, intestinal bacteria, clostridium perfringens, streptococcus aureus, mould, yeast.

8, method according to claim 1 is characterized in that a kind of general food inspection biochip, and all inspection for food hygiene standards are input in the computer, compares the standard of certain based food according to the detection data again and judges.