CN105177127A - Polynucleotide, method and kit for detecting listeria monocytogenes - Google Patents

Abstract

The invention discloses polynucleotide, a method and a kit for detecting listeria monocytogenes. Specifically, the invention discloses polynucleotide, which can be used as a standard molecule in realtime fluorescent PCR detection of listeria monocytogenes, and DNA constructs (LY01 and LY02). The provided plasmid standard molecule can solve the problem that realtime fluorescence PCR detection of listeria monocytogenes is lack of standard substances, and moreover, can guarantee the comparability of the detection results of realtime fluorescence PCR detection. A reliable quality control method is provided for the realtime fluorescence PCR detection of listeria monocytogenes.

Description

A set of polynucleotide, method and test kit detected for Listeria monocytogenes

Technical field

The present invention relates to the plasmid molecule of a set of technical field of bioengineering, be specifically related to a set of polynucleotide, method and the test kit that detect for Listeria monocytogenes.

Background technology

Listeria Monocytogenes (L.monocytogenes) belongs to listeria (Listeria).Listeria monocytogenes is the germ of uniquely causing a disease to people in listeria, a kind of important Zoonosis and food sanitation pathogenic bacteria, this bacterium causes the infection morbidity of people mainly through meat breast and goods thereof, humans and animals meningitis septicemia and pregnant woman's miscarriage etc. can be caused, therefore caused the attention of multinational relevant departments of the world.

The detection method of current Listeria Monocytogenes is divided into two classes, and a class is traditional cultivation and improves one's methods, and depend on biochemistry and Morphological Characteristics, sense cycle is longer, poor operability; One class is polymerase chain reaction (polymerasechainreaction, PCR) methods involving, comprise regular-PCR method and real time fluorescent PCR method, multiple target genes of other bacterium of listeria are mainly different from for Listeria Monocytogenes, select distinguished sequence, set up PCR and real time fluorescent PCR method.Listeria Monocytogenes PCR related detecting method development in recent years is rapid, due to the characteristic that it is quick, sensitive, special, becomes the reliable detection method of food borne pathogenic microorganism.

Plasmid DNA reference material is the recombinant plasmid molecule of a set of specific fragment containing testing goal gene, can as positive control in PCR qualitative detection, as the standard substance of quantitative analysis, the typical curve of quantitative analysis can be built in PCR quantitative analysis.Current plasmid DNA molecule obtains increasing further investigation and application as the reference material of gene test, but still blank for the plasmid DNA reference material of Listeria Monocytogenes real-time fluorescence PCR detection method.When actual Listeria Monocytogenes real-time fluorescence PCR, mostly what constituent parts used be plasmid DNA molecule that designed, designed builds is as standard substance, goal gene specific fragment is wherein different, lack unified valued methods simultaneously, plasmid DNA molecule definite value poor accuracy, cause the detected result very different between each laboratory, lack comparability.

Summary of the invention

The object of the present invention is to provide a set of plasmid control molecule and the application thereof that are applicable to the detection of Listeria monocytogenes real-time fluorescence quantitative PCR.

Another object of the present invention is to provide a set of plasmid control molecule and the application thereof that are applicable to the detection of pathotype Listeria monocytogenes real-time fluorescence quantitative PCR.

A first aspect of the present invention, provides the polynucleotide of a set of separation, and described polynucleotide comprise the internalization plain gene InlA gene order of Listeria monocytogenes and/or the transcriptional activation Function protein gene prfA gene order of Listeria monocytogenes.

In another preference, the gene order of described Listeria monocytogenes internalization plain gene InlA is selected from lower group:

The polynucleotide sequence of (a) sequence as shown in SEQIDNO.:3;

The polynucleotide sequence of homology >=95% (preferably >=98%) of sequence shown in (b) nucleotide sequence and SEQIDNO.:3;

20%-100% (the preferably 50%-100% that c () sequence is mated completely with polynucleotide shown in SEQIDNO.:3 or complete complementary and length are sequence length shown in SEQIDNO.:3, more preferably 80%-100%, 90%-100% best, as 95%) polynucleotide sequence;

The polynucleotide sequence of the polynucleotide sequence complementation d () is arbitrary with (a)-(c) described in.

In another preference, the transcriptional activation Function protein gene prfA gene order of described Listeria monocytogenes is selected from lower group:

The polynucleotide sequence of (a) sequence as shown in SEQIDNO.:1;

The polynucleotide sequence of homology >=95% (preferably >=98%) of sequence shown in (b) nucleotide sequence and SEQIDNO.:1;

20%-100% (the preferably 50%-100% that c () sequence is mated completely with polynucleotide shown in SEQIDNO.:1 or complete complementary and length are sequence length shown in SEQIDNO.:1, more preferably 80%-100%, 90%-100% best, as 95%) polynucleotide sequence;

The polynucleotide sequence of the polynucleotide sequence complementation d () is arbitrary with (a)-(c) described in.

In another preference, described polynucleotide contain the transcriptional activation Function protein gene prfA gene order of Listeria monocytogenes and the internalization plain gene InlA sequence of Listeria monocytogenes, and optionally connect the joint sequence of two kinds of gene orders.

In another preference, described polynucleotide sequence is as shown in SEQIDNO.:3.

In another preference, described polynucleotide sequence is as shown in SEQIDNO.:1.

A second aspect of the present invention, provides the DNA construction of a set of separation, comprises the polynucleotide described in first aspect present invention in described DNA construction, and optional sequence label, cleavage sequence, promoter sequence and/or carrier sequence.

In another preference, in described DNA construction, comprise the gene order of the transcriptional activation Function protein gene prfA gene order of Listeria monocytogenes and the internalization element InlA of Listeria monocytogenes.

In another preference, described DNA construction is linear DNA construction or cyclic DNA construction.

In another preference, described DNA construction is plasmid or expression vector.

In another preference, the standard molecule (plasmid control molecule) that described plasmid or expression vector detect as Listeria monocytogenes.

In another preference, the skeleton plasmid of described plasmid or expression vector is selected from lower group: pUC19, pUC18, pUC118, pUC119, pBlueScriptIISK and pGEM.

In another preference, the sequence of described plasmid is as shown in SEQIDNO:2 or 4.

A third aspect of the present invention, provides a set of test kit, and described test kit comprises the polynucleotide described in first aspect present invention or the DNA construction described in second aspect present invention.

In another preference, in described test kit, also comprise primer pair, the gene order of described primer pair specific amplification Listeria monocytogenes internalization plain gene InlA.

In another preference, the described primer pair of the gene order of specific amplification Listeria monocytogenes internalization plain gene InlA is selected from lower group:

Primer pair shown in SEQIDNO.:5 and SEQIDNO.:6;

Primer pair shown in SEQIDNO.:7 and SEQIDNO.:8;

Primer pair shown in SEQIDNO.:9 and SEQIDNO.:10; With

Primer pair shown in SEQIDNO.:11 and SEQIDNO.:12.

In another preference, in described test kit, also comprise primer pair, the transcriptional activation Function protein gene prfA gene order of described primer pair specific amplification Listeria monocytogenes.

In another preference, the primer pair of the transcriptional activation Function protein gene prfA gene order of specific amplification Listeria monocytogenes is selected from lower group:

Primer pair shown in SEQIDNO.:14 and SEQIDNO.:15;

Primer pair shown in SEQIDNO.:16 and SEQIDNO.:17; With

Primer pair shown in SEQIDNO.:18 and SEQIDNO.:19.

In another preference, also comprise the probe sequence being selected from lower group in described test kit, described probe sequence is as shown in SEQIDNO.:13.

In another preference, also comprise the probe sequence being selected from lower group in described test kit, described probe sequence is as shown in SEQIDNO.:20.

A fourth aspect of the present invention, provides the purposes of the DNA construction described in polynucleotide as described in the first aspect of the invention, second aspect present invention or the test kit described in third aspect present invention, it is characterized in that, for the detection of Listeria monocytogenes.

In another preference, described in be detected as non-diagnostic or therapeutic purpose.

In another preference, described in be detected as fluorescence quantitative PCR detection.

A fifth aspect of the present invention, provides a set of Listeria monocytogenes real-time fluorescence quantitative PCR detection method, and institute's accepted standard material is polynucleotide as described in the first aspect of the invention or the DNA construction described in second aspect present invention.

A sixth aspect of the present invention, provide a set of polynucleotide product, described product comprises:

(i) Listeria monocytogenes standard substance, described standard substance are selected from: the polynucleotide described in first aspect present invention or the DNA construction described in second aspect present invention;

(ii) primer pair of specific amplification Listeria monocytogenes sequence, described primer pair is:

The primer pair of the Sequence composition shown in SEQIDNO.:5 and SEQIDNO.:6; And/or

The primer pair of the Sequence composition shown in SEQIDNO.:14 and SEQIDNO.:15.

In another preference, described product is the combination (combination) of polynucleotide, and preferably described component (i) and (ii) are separately independently.

In another preference, described product is kit form.

A seventh aspect of the present invention, provides the preparation method of the plasmid control molecule of a set of Listeria monocytogenes, comprises the following steps:

1. the internalization element InlA gene order of synthetic Listeria monocytogenes and/or the transcriptional activation Function protein gene prfA gene order of Listeria monocytogenes, the transcriptional activation Function protein gene prfA gene order of described Listeria monocytogenes and described internalization element InlA expressing gene sequence are respectively as shown in SEQIDNO.:1 and SEQIDNO:3;

2. by step 1. gained gene order be cloned on cloning vector, obtain the plasmid control molecule of Listeria monocytogenes.

In another preference, described step 2. described cloning vector is pUC19, pUC18, pUC118, pUC119, pBlueScriptIISK or pGEM.

In another preference, described step 2. described cloning vector is pUC19.

Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.

Accompanying drawing explanation

Fig. 1 is the collection of illustrative plates of plasmid control molecule LY01 of the present invention.

Fig. 2 is gained plasmid control molecule LY01 Detection of Stability result figure of the present invention.

Fig. 3 is the real-time fluorescence PCR typical curve utilizing plasmid control molecule LY01 of the present invention to set up.

Fig. 4 is the collection of illustrative plates of plasmid control molecule LY02 of the present invention.

Fig. 5 is gained plasmid control molecule LY02 Detection of Stability result figure of the present invention.

Fig. 6 is the real-time fluorescence PCR typical curve utilizing plasmid control molecule LY02 of the present invention to set up.

Embodiment

The present inventor is by extensive and deep research, obtain one section and can be used in the polynucleotide sequence of Listeria monocytogenes PCR detection and primer pair with matching, experimental result shows, adopt suitable skeleton plasmid that described polynucleotide sequence is prepared as standard plasmid molecule, and coordinate primer pair of the present invention to carry out real-time PCR detection, there is splendid specificity and sensitivity, and have good stability.

Technical problem to be solved by this invention is to overcome in existing Listeria monocytogenes real-time fluorescence PCR detection method the problem lacking positive criteria product and the configuration of positive criteria product, provide a set of be applicable to Listeria monocytogenes real-time PCR detection plasmid control molecule and the construction process of this plasmid control molecule, quantivative approach and application.

Detect bacterial strain

Listeria monocytogenes (L.monocytogenes, LM) be pathogenic bacteria important in food sanitation, it is extensively present in nature, humans and animals meningitis miscarriage septicemia etc. can be caused, case fatality rate is up to 30% ~ 70%, be one of the current mankind most important borne Parasitic Encephalopathy cause of disease bacterium, WHO is classified as one of four large pathogenic bacterium in the food nineties in 20th century, is also listed in 21 century to one of Chinese's hygiene and health 12 kinds of pathogenic micro-organisms with great effect.

Listeria monocytogenes transcriptional activation Function protein gene prfA gene (corresponding detection plasmid control material LY02) is the virulence factor that Listeria monocytogenes detects.Research data shows, the Inl gene of coding internalization element is the most important Disease-causing gene of LM, and its site is in the karyomit(e) of Listeria monocytogenes, and lack i.e. no pathogenicity, therefore, they can as the key gene of Listeria monocytogenes identification.

The principle of real-time PCR detection Listeria monocytogenes

Adopt real-time fluorescence PCR technology can internalization element InlA gene order or Listeria monocytogenes transcriptional activation Function protein gene prfA gene order in specific amplification Listeria monocytogenes genomic dna, design for the primer of above target gene and the probe of two ends mark fluorescent, amplification assay sample DNA.Real-time fluorescence PCR can carry out Real-Time Monitoring PCR primer by the increase detecting fluorescent signal.Meanwhile, with the positive criteria material (or positive criteria molecule) of identical primer, probe and condition amplification concentration known.In PCR method, positive criteria material (or positive criteria molecule) can as positive control; In real-time fluorescence PCR, positive criteria material (or positive criteria molecule) can build stable typical curve, can calculate the absolute content (copy number or concentration) of corresponding gene in sample according to typical curve respectively.

Reference material

Reference material has a set of or multiple enough all even characteristic value determined very well, in order to correcting device, evaluates measuring method or to the material of material assignment or material.

Plasmid control molecule

Relate to two kinds in the present invention detect the specific sequence of Listeria monocytogenes and devise two kinds of plasmid control molecules on this basis, a kind of specific sequence of Listeria monocytogenes is Listeria monocytogenes internalization element InlA gene, and length is 2403bp; The specific sequence of another set of Listeria monocytogenes is Listeria monocytogenes transcriptional activation Function protein gene prfA gene, and length is 714bp.

At one preferably in embodiment of the present invention, the invention provides the plasmid control molecule of a set of Listeria monocytogenes, this plasmid control molecule comprises Listeria monocytogenes transcriptional activation Function protein gene prfA gene order, and described Listeria monocytogenes transcriptional activation Function protein gene prfA gene order is as shown in SEQ ID NO:1:

Plasmid control molecule of the present invention, the target gene Listeria monocytogenes transcriptional activation Function protein gene prfA gene order that the PCR preferably containing Listeria monocytogenes detects.

Listeria monocytogenes transcriptional activation Function protein gene prfA gene described in the present invention, work in the synthesis of DNA, RNA and protein, it can be used as a set of target gene that Listeria monocytogenes detects, and the sequence of described Listeria monocytogenes transcriptional activation Function protein gene prfA gene is preferably as shown in SEQIDNO:1.

The sequence of plasmid control molecule of the present invention is preferably as shown in SEQIDNO:2, and wherein the 414th is prfA gene order to the 1127th:

Of the present invention another preferably in embodiment, the invention provides the plasmid control molecule of a set of Listeria monocytogenes, this plasmid control molecule comprises Listeria monocytogenes internalization element InlA expressing gene sequence, and described Listeria monocytogenes internalization element InlA expressing gene sequence is as shown in SEQ ID NO:3:

Plasmid control molecule of the present invention, the target gene Listeria monocytogenes internalization element InlA expressing gene sequence that the PCR preferably containing Listeria monocytogenes detects.

Listeria monocytogenes internalization element InlA expressing gene described in the present invention, work in the synthesis of DNA, RNA and protein, it can be used as a set of target gene that Listeria monocytogenes detects, and the sequence of described Listeria monocytogenes internalization element InlA is preferably as shown in SEQIDNO:3.

Of the present invention another preferably in embodiment, the sequence of plasmid control molecule of the present invention is as shown in SEQIDNO:4, and wherein the 414th is InlA gene order to the 2816th:

Of the present invention another preferably in embodiment, according in plasmid control molecule of the present invention containing Listeria monocytogenes transcriptional activation Function protein gene prfA gene order and Listeria monocytogenes internalization element InlA gene order.Preferably, described prfA sequence is as shown in SEQIDNO.:1, and described InlA sequence is as shown in SEQIDNO.:3.

The quantivative approach of the plasmid control molecule of Listeria monocytogenes described above in the present invention, it comprises the following steps:

1. plasmid control molecule is extracted;

2. according to the step 1. based composition of the plasmid control molecule of gained and sequence length, the content of the phosphoric in plasmid control molecule is calculated;

3. prepare the phosphorus standardized solution of gradient concentration, and make high resolution inductively coupled plasma with this standardized solution and launch mass spectrographic typical curve;

4. high resolution inductively coupled plasma launches mass spectrometric detection plasmid control molecule solution, and draws the phosphorus content of plasmid control molecule solution according to the typical curve of step 3. gained;

5. according to the content of the phosphoric in the plasmid control molecule of the step 4. phosphorus content of the plasmid control molecule solution of gained and step 2. gained, the concentration of plasmid control molecule is calculated.

Wherein described in step, 3. the condition of high resolution inductively coupled plasma transmitting mass spectrometric detection is as follows: cooling gas flow is preferably 10L/min ~ 25L/min, be 16.86L/min best, assisted gas flow is preferably 0.5L/min ~ 3.0L/min, be 0.88L/min best, atomization gas flow is be preferably 0.5L/min ~ 2.43L/min, be more preferably 1.123L/min, power is preferably 1200W ~ 1400W, is 1350W best.

The quantivative approach of plasmid control molecule of the present invention preferably comprises ultraviolet spectrophotometry and High resolution-inductive coupled plasma mass spectrometry (HR-ICP-MS).

Concrete implementation:

The quantivative approach of plasmid control molecule of the present invention preferably comprises ultraviolet spectrophotometry and High resolution-inductive coupled plasma mass spectrometry (HR-ICP-MS).

Wherein ultraviolet spectrophotometry preferably comprises the following steps plasmid control molecule quantivative approach:

1. plasmid control molecule is extracted;

2. ultraviolet spectrophotometer is made to be corrected to zero point with TE damping fluid;

3. appropriate DNA (needs according to instrument) is got to (nanodrop directly puts at surveyed area) in cuvette, recording instrumnet reading.

If 4. can directly read DNA concentration, directly record; If can not, then record the optical density(OD) of sample at 260nm and 280nm, the concentration of DNA sample is OD260 × nucleic acid extension rate × 50, and concentration unit is ng/ μ L.

(2) HR-ICP-MS preferably comprises the following steps plasmid control molecule quantivative approach:

1. plasmid control molecule is extracted;

2. according to based composition and the sequence length of plasmid control molecule, the content of the phosphoric of each plasmid control molecule is calculated respectively;

3. prepare the phosphorus standardized solution of gradient concentration, and make the typical curve of HR-ICP-MS with this standardized solution.

4. HR-ICP-MS detects plasmid control molecule, and draws the phosphorus content of plasmid control molecule according to typical curve.

5. the concentration of plasmid control molecule is calculated according to the phosphorus content of plasmid control molecule.

Present invention also offers a set of Listeria monocytogenes real-time fluorescence quantitative PCR detection method, its accepted standard material is plasmid control molecule as above.

Present invention also offers the preparation method of the plasmid control molecule of a set of Listeria monocytogenes as above, comprise the following steps:

1. the specific sequence of Listeria monocytogenes described in synthetic, described sequence is as shown in SEQIDNO:1 and/or 3;

2. by the sequence clone of step 1. gained Listeria monocytogenes on cloning vector, obtain the plasmid control molecule of Listeria monocytogenes.

Wherein the method for the synthetic that step is 1. described is preferably: the method for full genome synthesis or PCR primer amplification obtains this sequence.

Plasmid control molecule construction process of the present invention preferably comprises the following steps:

In the Genbank of 1. NCBI (US National Biotechnology Information center), cargo tracer increases listerial internalization plain gene InlA gene and/or transcriptional activation Function protein gene prfA gene;

2. above-mentioned sequence is analyzed, select suitable sequence and suitable restriction enzyme site, and restriction enzyme site is added to 5 ' end and the 3 ' end of selected sequence.

3. the sequence after process is carried out the service of full genome synthetic, comprise the work such as the synthesis of strand OligoDNA, DNA fragmentation splicing, and full-length gene is cloned in plasmid vector, obtain plasmid control molecule.

Described plasmid vector can be conventional carrier, preferably cloning vector, the cloning vector more preferably can bred in intestinal bacteria, described cloning vector is preferably: pUC19, pUC18, pUC118, pUC119, pBlueScriptIISK or pGEM serial carrier, is preferably pUC19 cloning vector.

4. the sequence of sequence verification plasmid control molecule.

5. the real-time fluorescence PCR detection method checking of plasmid control molecule.

Described real-time fluorescence PCR detection method checking, refer to and detect the specificity of plasmid control molecule when carrying out real-time fluorescence PCR and analyzing and build the characteristics such as typical curve ability, to identify the ability of this plasmid control molecule as the reference material of real time fluorescent PCR method detection Listeria monocytogenes.

On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.

Agents useful for same of the present invention and raw material are all commercially.

Major advantage of the present invention is:

(1) it is strong that the plasmid control molecule comprising polynucleotide sequence of the present invention has homogeneity, the advantage that stability is high, the invention solves a difficult problem for Listeria monocytogenes real-time PCR detection Plays material want simultaneously, ensure the comparability of Listeria monocytogenes real time fluorescent PCR method detected result, provide quality control for Listeria monocytogenes real time fluorescent PCR method detects;

(2) product using plasmid control molecule of the present invention to coordinate primer pair of the present invention to prepare, during for real-time PCR detection Listeria monocytogenes, high specificity, highly sensitive, linear stable.

(3) plasmid control molecule (carrying the LY02 plasmid control molecule of transcriptional activation Function protein gene prfA gene) causing plasmid control molecule (carrying the LY01 plasmid control molecule of internalization element InlA gene) and/or detection pathotype Listeria monocytogenes detecting Listeria monocytogenes is contained in test kit provided by the invention

Below in conjunction with specific embodiment, state the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted detailed conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.

The structure of embodiment 1 plasmid control molecule

Experiment reagent and laboratory apparatus:

Plasmid extracts test kit (OMEGA) in a large number, and other biochemical reagents are import packing or domestic analytical pure biochemical reagents; Laboratory apparatus comprises whizzer, thermostat water bath, constant-temperature shaking incubator, liquid-transfering gun etc.

Experimental technique comprises the following steps:

1, in GenBank, search for the gene internalization element InlA gene order of Listeria monocytogenes;

2, analyze above-mentioned sequence, select suitable sequence and suitable restriction enzyme site, gene internalization element InlA gene order length is 2403bp, and two ends add KpnI and BamHI restriction enzyme site;

3, sequence after process is delivered to precious biotechnology (Dalian) company limited, be responsible for carrying out the service of full genome synthetic by it, comprise the synthesis of strand OligoDNA, the work such as DNA fragmentation splicing, the sequence of gained gene internalization element InlA gene is as shown in SEQ ID NO:3, gained full-length gene is cloned in plasmid vector pUC19 (purchased from TAKARA company), build the plasmid control molecule LY01 (its sequence is as shown in SEQ ID NO:4) obtaining and comprise OMPW gene order, build the plasmid map of gained plasmid control molecule LY01 as shown in Figure 1.

4, mass propgation contains the recombination bacillus coli comprising gained plasmid control molecule LY01, utilizes plasmid to extract test kit (OMEGA) in a large number and extracts plasmid, obtain highly purified plasmid control molecule LY01.Carry out purity check through ultraviolet spectrophotometer and electrophoresis, plasmid DNA standard molecule is placed in-20 DEG C of preservations afterwards, and the method for extracting plasmid comprises the following steps:

A, by the bacterium of 100 ~ 200mL incubated overnight in room temperature 5000 × g centrifugal 10 minutes, supernatant discarded;

B, add 12mLSolution I (containing RNaseA), vibration fully mixes;

C, add 12mLSolution II, gentle mixing of turning upside down, room temperature places 2 minutes to make the abundant cracking of thalline;

D, add 16mLSolution III, fully put upside down mixing immediately up and down for several times, until form uniform white precipitate;

E, >=12000 × g, 4 DEG C are centrifugal 10 minutes;

F, draw 20mL supernatant and move in a clean HiBindMaxi adsorption column (being placed in 50mL collection tube) carefully, centrifugal 5 minutes of 5000 × g room temperature;

G, the liquid discarded in collection tube, add 10mLBufferHB and clean adsorption column, centrifugal 5 minutes of 5000 × g room temperature;

H, the liquid discarded in collection tube, add 15mLDNAWashBuffer (dehydrated alcohol dilution) and clean adsorption column;

I, repeat above-mentioned cleaning step;

Centrifugal 15 minutes of j, 6000 × g are with dry adsorption column;

K, adsorption column is placed in a clean 50mL pipe, add 2mL ~ 3mLTE damping fluid, room temperature is placed 1 ~ 2 minute, and within centrifugal 2 minutes, with eluted dna, gained DNA solution is the plasmid control molecule solution of extraction to 8000 × g, is kept at-20 DEG C.

5, sequence verification plasmid control molecule LY01

The plasmid control molecule of extraction is delivered to eight order-checking companies and carry out sequence verification, eight order-checking companies are respectively Beijing Liuhe Huada Genomics Technology Co., Ltd, Shanghai Bo Shang Bioisystech Co., Ltd, prompt base (Shanghai) trade Co., Ltd in the English Weihe River, Shanghai Jie Li Bioisystech Co., Ltd, Shanghai Mei Ji Bioisystech Co., Ltd, Shanghai Sheng Gong Bioisystech Co., Ltd, Jin Weizhi bio tech ltd, Suzhou, precious biotinylated biomolecule Engineering Co., Ltd.For plasmid DNA reference material, sequence length with quantitatively have direct relation, it is requirement for definite value in Developments of certified reference samples that 8 different order-checking companies carry out sequencing.Refer to ISO directive/guide 35, the specific requirement of reference material definite value part.

Sequence investigates formula: sequence accuracy=correct base number/base sum

Sequencing result proves, the sequence accuracy that the unit of all participation sequence verification obtains is 100%, and the 414th in this plasmid sequence to the 2816th is gene internalization element InlA gene.

Experimental result is: obtain highly purified plasmid control molecule LY01 through sequence verification through steps such as sequence selection step, full genome synthetic step and plasmid extract in a large number, and this plasmid control molecule Insert Fragment sequence conforms to completely with design.

Adopt above-mentioned identical method, build LY02 plasmid, by gained gene transcriptional activation Function protein gene prfA gene order (as shown in SEQ ID NO:1), be cloned in plasmid vector pUC19, build the plasmid control molecule LY02 (its sequence is as shown in SEQ ID NO:2) obtaining and comprise prfA gene order, build the plasmid map of gained plasmid control molecule LY02 as shown in Figure 4.Through sequence verification, this plasmid control molecule Insert Fragment sequence conforms to completely with design.

The uniformity testing of embodiment 2 plasmid control molecule LY01 and LY02

Homogeneity is the coherency state characterizing structure that a set of in material or multifrequency nature is correlated with or composition.By measuring the sample of the prescribed level taking from Different Package unit (as bottle, bag etc.) or take from same packaging unit different positions, measuring result drops in regulation range of uncertainty, then can think that this reference material is uniform to the characteristic quantity of specifying.Homogeneity is the base attribute of reference material, for the spatial distribution characteristic of description standard substance characteristics.Homogeneity assessment must be carried out, to prove that it has good homogeneity in development (production) process of reference material.The plasmid control molecule had good uniformity, its value can not be subject to the impact of the factors such as packing, and the value difference between each bottle is little, therefore ensure that the reliability of detected result.

Experiment reagent TE damping fluid (pH7.5).Laboratory apparatus NanodropsND-2000.

Experimental technique:

1, according to " JJG1006-1994 primary standard material technical specifications " homogeneity draw samples requirement, from each plasmid DNA reference material, 15 bottles are randomly drawed.

2, to got each sample 3 times, each sampling amount is 1 μ L.Each sampling amount uv-spectrophotometric replication 3 times, averages.

3, measurement result F method of inspection is added up, judge uniformity testing result.Concrete grammar is: extract m sample, records m group equally accurate measurement data under the same conditions, if measure variance there was no significant difference, then should meet the statistical requirements of following formula.

$F=\frac{\frac{Q_1}{v_1}}{\frac{Q_2}{v_2}}\≤F_{\mathrm{\α}}({\mathrm{\ν}}_1,{\mathrm{\ν}}_2)$

Wherein between group, sum of squares of deviations calculation formula is:

$Q_1=\underset{i=1}{\overset{m}{\Σ}}{n}_i{(\stackrel{\‾}{x_i}-\stackrel{\‾}{\stackrel{\‾}{x}})}^2$

In group, the sum of squares of deviations calculates and sees that formula is:

$Q_2=\underset{i=1}{\overset{m}{\Σ}}\underset{j=1}{\overset{nj}{\Σ}}{(x_{ij}-\stackrel{\‾}{x_i})}^2$

ν ₁=m-1 (between group degree of freedom)

ν ₂=N-m (the group internal degree of freedom).

Experimental result:

1, the uniformity testing result of plasmid control molecule LY01 and LY02 is respectively as shown in table 1-4

Table 1 plasmid control molecule LY01 uniformity testing data (unit: ng/ μ L)

The homogeneity statistical result of table 2 plasmid control molecule LY01

The homogeneity statistical of LY01 the results are shown in Table 2, and under 95% confidence level, F value is less than F _0.05(14,30), prove that this plasmid control molecule LY01 is uniform, and be evenly up to the standards requirement to meet " JJG1006-94 primary standard material technical specifications ".

Table 3 plasmid control molecule LY02 uniformity testing data (unit: ng/ μ L)

The homogeneity statistical result of table 4 plasmid control molecule LY02

The homogeneity statistical of LY02 the results are shown in Table 3, and under 95% confidence level, F value is less than F _0.05(14,30), prove that this plasmid control molecule LY02 is uniform, and be evenly up to the standards requirement to meet " JJG1006-94 primary standard material technical specifications ".

The study on the stability of embodiment 3 plasmid control molecule LY01 and LY02

Stability refers under the specific timed interval and storage requirement, and the characteristic value of reference material remains on the ability in specialized range.Stability is the base attribute of reference material, the time dependent character of the characteristic for description standard material, the i.e. Time-distribution of description standard substance characteristics.Stability assessment must be carried out in the triturating of reference material.Stability assessment not only can assess the uncertainty of measurement relevant to stability of material, and can specify suitable storage and transport condition.The plasmid control molecule had good stability, As time goes on its characteristic value can not change under suitable storage and transport condition, and detected result instablely can not to affect by it, ensure that the reliability of detected result.

Experiment reagent TE damping fluid (pH7.5).Laboratory apparatus NanodropsND-2000.

Experimental technique:

Adopt the stability of classical stability study to plasmid control molecule to investigate, As time goes on the sample namely simultaneously prepared is measured under the same conditions.

1,3 bottles were randomly drawed to the plasmid control molecule (-20 DEG C of preservations) of preparation in 0th month after prepared by plasmid control molecule, 0.5 month, 1 month, 2 months, 4 months, 7 months, 10 months, 12 months, each sample replication 3 times, average, carry out permanent stability investigation.This research have employed ultraviolet spectrophotometry and has carried out following the tracks of investigating to plasmid control molecule.

2, Detection of Stability data are assessed, judge study on the stability result.Concrete investigation method is as follows:

Straight slope can calculate with following formula:

${\mathrm{\β}}_1=\frac{\underset{i=1}{\overset{n}{\Σ}}(X_i-\stackrel{\‾}{X})(Y_i-\stackrel{\‾}{Y})}{\underset{i=1}{\overset{n}{\Σ}}{(X_i-\stackrel{\‾}{X})}^2}$

In formula: X _i---i-th time point; Y _i---the observed value of i-th time point; ---the mean value of all time points; ---the mean value of all observed values.

Intercept can be calculated by following formula:

${\mathrm{\β}}_0=\stackrel{\‾}{Y}-{\mathrm{\β}}_1\stackrel{\‾}{X}$

On straight line, the standard deviation of often can calculate with following formula:

$s^2=\frac{\underset{i=1}{\overset{n}{\Σ}}{(Y_i-{\mathrm{\β}}_0-{\mathrm{\β}}_1{X}_i)}^2}{n-2}$

In formula: X _i---i-th time point; Y _i---the observed value of i-th time point; β ₁, β ₀---regression coefficient; N---measurement coefficient.

β ₁standard deviation provided by following formula:

$s\left({\mathrm{\β}}_1\right)=\frac{s}{\sqrt{\underset{i=1}{\overset{n}{\Σ}}{(X_i-\stackrel{\‾}{X})}^2}}$

Based on β ₁standard deviation, available t-detects and carries out following judgement: even | β ₁| < t _{0.95, n-2}s (β ₁), then show that slope is not remarkable, do not observe unstable.

Experimental result:

1, plasmid control molecule LY01 study on the stability result is as shown in table 5 and Fig. 2:

The STABILITY MONITORING data (unit: ng/ μ L) of table 5 plasmid control molecule LY01

Statistics is as shown in table 6.

The stability statistics of table 6 plasmid control molecule LY01

From statistics, it 12 months is stable that plasmid control molecule LY01 preserves under-20 degree conditions.

2, plasmid control molecule LY02 study on the stability result is as shown in table 7 and Fig. 5:

The STABILITY MONITORING data (unit: ng/ μ L) of table 7 plasmid control molecule LY02

Statistics is as shown in table 8.

The stability statistics of table 8 plasmid control molecule LY02

From statistics, it 12 months is stable that plasmid control molecule LY02 preserves under-20 DEG C of conditions.

Experimental example 4 ultraviolet spectrophotometry is carried out quantitatively plasmid control molecule

Experiment reagent TE damping fluid (pH7.5).Laboratory apparatus NanodropsND-2000.

Experimental technique comprises the following steps:

1, ultraviolet spectrophotometer is made to be corrected to zero point with TE damping fluid;

2, Example prepares gained DNA solution 1 μ L, directly at surveyed area, and recording instrumnet reading;

3, directly read DNA concentration, concentration unit is ng/ μ L.

4, each sample test eight times, averages.

Experimental result:

1, through ultraviolet spectrophotometry definite value, the concentration of plasmid control molecule LY01 is as shown in table 9:

Table 9 ultraviolet spectrophotometry definite value result (unit: ng/ μ L)

Plasmid	Repeat 1	2	3	4	5	6	7	8	Mean value	SD
											LY01	92.4	92.7	93.3	93.4	93.6	93.6	93.8	94	93.4	0.55

2, through ultraviolet spectrophotometry definite value, the concentration of plasmid control molecule LY02 is as shown in table 5:

Table 10 ultraviolet spectrophotometry definite value result (unit: ng/ μ L)

Plasmid	Repeat 1	2	3	4	5	6	7	8	Mean value	SD
											LY02	57.1	56.2	59.3	56.5	57.6	59.7	58.5	59.5	58.1	1.39

Embodiment 5 HR-ICP-MS carries out quantitatively plasmid control molecule LY01 and LY02

Experiment reagent is P standardized solution (NIST, SRM3139a).Laboratory apparatus is: inductively coupled plasma launches mass spectrograph (Thermofisherelement2).

Experimental technique comprises the following steps:

1, according to based composition and the sequence length of plasmid control molecule, the content of wherein phosphoric is calculated respectively;

2, HR-ICP-MS experiment parameter is: cooling gas flow 16.86L/min, and atomization gas flow is 1.123L/min, and assisted gas flow is 0.99L/min, power 1350W.

3, prepare gradient concentration phosphorus standardized solution (0,1,2,3,4,5g/L), and make the typical curve of HR-ICP-MS with this standardized solution;

4, plasmid control molecule is diluted 3000 times, HR-ICP-MS detects the plasmid control molecule after dilution, and draws the phosphorus content of the plasmid control molecule after dilution according to typical curve;

5, the concentration of plasmid control molecule is calculated according to the phosphorus content recorded and extension rate;

6, each sample test eight times, averages.

Experimental result:

1, as calculated, the content of the phosphoric of plasmid control molecule LY01 is 10.21%.Obtain plasmid control molecule phosphorus element content data through HR-ICP-MS definite value as shown in table 11, extension rate is 3000, converts and obtains the mass concentration of plasmid molecule, as shown in table 12.

The phosphorus element content (unit: μ g/L) of table 11 plasmid control molecule LY01

The mass concentration result (unit: ng/ μ L) of table 12 plasmid control molecule LY01

2, as calculated, the content of the phosphoric of plasmid control molecule LY02 is 10.22%.Obtain plasmid control molecule phosphorus element content data through HR-ICP-MS definite value as shown in table 13, extension rate is 2000, converts and obtains the mass concentration of plasmid molecule, as shown in table 14.

The phosphorus element content (unit: μ g/L) of table 13 plasmid control molecule LY02

Plasmid	Repeat 1	2	3	4	5	6	7	8	Mean value	SD
											LY02	3.07	3.02	3.07	2.93	2.87	2.92	2.82	2.85	2.96	0.09

The mass concentration result (unit: ng/ μ L) of table 14 plasmid control molecule LY02

The application of plasmid control molecule LY01 and LY02 in real-time PCR detection that embodiment 6 is developed

Experiment reagent: for LY01 and LY02 plasmid control molecule, devise tens of to primer, the gene fragment increased in target sequence respectively, the primer of design and probe are transferred to precious biotechnology (Dalian) company limited to synthesize, MasterMix is purchased from Lifetechnology company.Laboratory apparatus comprises: real-time fluorescence quantitative PCR amplification instrument (Lifetechnology) whizzer, thermostat water bath, incubator, sky equality.

The application of plasmid control molecule LY01 and LY02 in real-time PCR detection:

Primer (using primer shown in table 15 respectively), probe, PCR detection system

PCR application of sample system:

Reaction conditions PCR program:

95 DEG C 10 minutes;

95 DEG C 15 seconds, 60 DEG C 1 minute, 40 circulations.

The plasmid control molecule (1.85 × 10 that the plasmid control molecule LY01 dilution of gained is different concns prepared by embodiment 1 ⁶copies/ μ L, 1.85 × 10 ⁵copies/ μ L, 1.85 × 10 ⁴copies/ μ L, 1.85 × 10 ³copies/ μ L, 1.85 × 10 ²copies/ μ L, 1.85 × 10 ¹copies/ μ L, 1.85 × 10 ⁰copies/ μ L), LY02 dilution is the plasmid control molecule (1.69 × 10 of different concns ⁶copies/ μ L, 1.69 × 10 ⁵copies/ μ L, 1.69 × 10 ⁴copies/ μ L, 1.69 × 10 ³copies/ μ L, 1.69 × 10 ²copies/ μ L, 1.69 × 10 ¹copies/ μ L, 1.69 × 10 ⁰copies/ μ L) using the plasmid control molecule of different concns as template, carry out real-time fluorescent PCR amplification according to the method in document.Each reaction in triplicate, according to the relation between the Ct value of different concns template amplification and concentration, Criterion curve.

Experimental result

1, to tens of, primer is tested in this example, experimental result shows have 4 pairs of primers effectively can increase for the gene order of the internalization element InlA of Listeria monocytogenes, has 3 pairs of primers effectively can increase for the transcriptional activation Function protein gene prfA gene order of Listeria monocytogenes.Final acquisition the primer of Successful amplification target sequence and probe sequence can refer to table 15.

The primer that table 15 adopts in testing and probe sequence

For the gene order of the internalization element InlA of Listeria monocytogenes, the Detection results of primer pair 1 is best, high specificity, and do not have nonspecific band to occur after amplification, detection sensitivity is the highest, the minimum target sequence detecting 1.85copies/ μ L; The sensitivity of primer pair 2 and 3 is lower, can detect 1.85 × 10 ²the target sequence of copies/ μ L; Primer pair 4 can detect 1.85 × 10 ¹the target sequence of copies/ μ L, but specificity is poor, has non-specific band to occur.

For the transcriptional activation Function protein gene prfA gene order of Listeria monocytogenes, the Detection results of primer pair 5 is best, high specificity, does not have nonspecific band to occur after amplification, detection sensitivity is the highest, the minimum target sequence detecting 1.69copies/ μ L; The sensitivity of primer pair 6 is lower, can detect 1.69 × 10 ²the target sequence of copies/ μ L; Primer pair 7 can detect 1.96 × 10 ¹the target sequence of copies/ μ L, but specificity is poor, has non-specific band to occur.

2, the application of plasmid control molecule LY01 in real-time PCR detection

Use primer pair 1, respectively with the plasmid control molecule LY01 (as: 1.85 × 10 of different concns ⁶copies/ μ L, 1.85 × 10 ⁵copies/ μ L, 1.85 × 10 ⁴copies/ μ L, 1.85 × 10 ³copies/ μ L, 1.85 × 10 ²copies/ μ L, 1.85 × 10 ¹copies/ μ L, 1.85 × 10 ⁰copies/ μ L) typical curve of real-time fluorescence PCR is set up as standard substance.The typical curve set up is shown in Fig. 3, and relation conefficient reaches 0.999, linearly well, shows that plasmid control molecule LY01 is applicable to being applied to real-time PCR detection, can be used as the positive criteria material of real-time fluorescent PCR amplification Listeria monocytogenes.

Use primer pair 5, respectively with the plasmid control molecule LY02 (as: 1.69 × 10 of different concns ⁶copies/ μ L, 1.69 × 10 ⁵copies/ μ L, 1.69 × 10 ⁴copies/ μ L, 1.69 × 10 ³copies/ μ L, 1.69 × 10 ²copies/ μ L, 1.69 × 10 ¹copies/ μ L, 1.69 × 10 ⁰copies/ μ L) typical curve of real-time fluorescence PCR is set up as standard substance.The typical curve set up is shown in Fig. 6, and relation conefficient reaches 0.998, linearly well, shows that plasmid control molecule LY02 is applicable to being applied to real-time PCR detection, can be used as the positive criteria material of real-time fluorescent PCR amplification Listeria monocytogenes.

When actual Listeria monocytogenes detects, the typical curve that this linear good primer pair makes can be utilized, fluorescence PCR method detects the copy number of Listeria monocytogenes specific gene in testing sample by experiment, and according to the linear relationship between the copy number of Listeria monocytogenes specific gene and total plate count, the concrete number of Listeria monocytogenes can be conversed.Because the research and development of the current reference material for Listeria monocytogenes real-time fluorescence PCR detection method are still blank, when actual Listeria monocytogenes PCR in real time detects, mostly what constituent parts used be plasmid DNA molecule that designed, designed builds is as standard substance, goal gene specific fragment is wherein different, lack unified valued methods simultaneously, plasmid DNA molecule definite value poor accuracy, causes the detected result very different between each laboratory, lacks comparability, validity and reliability.Therefore, this plasmid control molecule lacks a difficult problem for reference material when can solve real-time PCR detection Listeria monocytogenes, be applicable to the real time fluorescent PCR method of multiple amplification Listeria monocytogenes, ensure the comparability of detected result, provide biometric technology support for Listeria monocytogenes real time fluorescent PCR method detects.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (10)

1. the polynucleotide be separated, it is characterized in that, the sequence of described polynucleotide is selected from lower group:

The polynucleotide sequence of (a) sequence as shown in SEQIDNO.:3 or 1;

The polynucleotide sequence of homology >=95% (preferably >=98%) of sequence shown in (b) nucleotide sequence and SEQIDNO.:3 or 1;

20%-100% (the preferably 50%-100% that c polynucleotide shown in () sequence with SEQIDNO.:3 or 1 mate completely or complete complementary and length are sequence length shown in SEQIDNO.:3 or 1, more preferably 80%-100%, 90%-100% best, as 95%) polynucleotide sequence;

The polynucleotide sequence of the polynucleotide sequence complementation d () is arbitrary with (a)-(c) described in.

2. the DNA construction be separated, is characterized in that, comprise polynucleotide according to claim 1 in described DNA construction, and optional sequence label, cleavage sequence, promoter sequence and/or carrier sequence.

3. DNA construction as claimed in claim 2, it is characterized in that, described DNA construction is linear DNA construction or cyclic DNA construction; Preferably described DNA construction is plasmid or expression vector; More preferably, the sequence of described plasmid or expression vector is as shown in SEQIDNO:4 or 2.

4. a test kit, is characterized in that, described test kit comprises polynucleotide described in claim 1 or DNA construction according to claim 2.

5. test kit as claimed in claim 4, it is characterized in that, also primer pair is comprised, the described gene order of primer pair specific amplification Listeria monocytogenes internalization plain gene InlA and/or the transcriptional activation Function protein gene prfA gene order of specific amplification Listeria monocytogenes in described test kit; Preferably the described primer pair of the gene order of specific amplification Listeria monocytogenes internalization plain gene InlA is selected from lower group:

Primer pair shown in SEQIDNO.:5 and SEQIDNO.:6;

Primer pair shown in SEQIDNO.:7 and SEQIDNO.:8;

Primer pair shown in SEQIDNO.:9 and SEQIDNO.:10; With

Primer pair shown in SEQIDNO.:11 and SEQIDNO.:12;

The primer pair of the transcriptional activation Function protein gene prfA gene order of specific amplification Listeria monocytogenes is selected from lower group:

Primer pair shown in SEQIDNO.:14 and SEQIDNO.:15;

Primer pair shown in SEQIDNO.:16 and SEQIDNO.:17; With

Primer pair shown in SEQIDNO.:18 and SEQIDNO.:19.

6. the purposes of polynucleotide, DNA construction according to claim 2 or test kit according to claim 4 as claimed in claim 1, is characterized in that, for the detection of Listeria monocytogenes.

7. a Listeria monocytogenes real-time fluorescence quantitative PCR detection method, is characterized in that, institute's accepted standard material is polynucleotide as claimed in claim 1 or DNA construction according to claim 2.

8. a polynucleotide product, is characterized in that, described product comprises:

(i) Listeria monocytogenes standard substance, described standard substance are selected from: DNA construction described in polynucleotide described in claim 1 or claim 2;

(ii) primer pair of specific amplification Listeria monocytogenes sequence, described primer pair is:

The primer pair of the Sequence composition shown in SEQIDNO.:5 and SEQIDNO.:6; And/or

The primer pair of the Sequence composition shown in SEQIDNO.:14 and SEQIDNO.:15.

9. a preparation method for the plasmid control molecule of Listeria monocytogenes, is characterized in that, comprises the following steps:

1. the plain gene order of InlA of the internalization of synthetic Listeria monocytogenes and/or the transcriptional activation Function protein gene prfA gene order of Listeria monocytogenes, the gene order of the transcriptional activation Function protein gene prfA gene order of described Listeria monocytogenes and the internalization element InlA of described Listeria monocytogenes is respectively as shown in SEQIDNO.:1 and SEQIDNO:3;

2. by step 1. gained gene order be cloned on cloning vector, obtain the plasmid control molecule of Listeria monocytogenes.

10. the preparation method of the plasmid control molecule of Listeria monocytogenes as claimed in claim 9, it is characterized in that, step 2. described cloning vector is pUC19, pUC18, pUC118, pUC119, pBlueScriptIISK or pGEM.