CN105112406A - Nucleotides specific to Hafnia alveibifermentans G58097, G5898 and G5900 and their application - Google Patents

Abstract

The invention relates to nucleotides specific to Hafnia alveibifermentans G58097, G5898 and G5900 and their application. The nucleotides include one type of nucleotides shown in SEQ ID NO: 1 to 6. These nucleotides are applicable to the preparation of PCR kits and gene chips used for detecting Hafnia alveibifermentans. The invention relates to the nucleotides specific to Hafnia alveibifermentans G58097, G5898 and G5900, and a PCR kit and a gene chip both including the nucleotides; an operating method is simple, a detection period is short, detection cost is lower, accuracy is high, sensitivity is high, and the nucleotides, the PCR kit and the gene chip are easy to industrially produce.

Description

To hafnia alvei G5897, the Nucleotide that G5898, G5900 are special and application thereof

Technical field

The present invention relates to hafnia alvei G5897, the Nucleotide that G5898, G5900 are special, particularly relate to hafnia alvei G5897, the Nucleotide that in the O antigen gene cluster of G5898, G5900, individual gene is special and application thereof.

Background technology

Hafnia alvei (Hafniaalvei) is the one in enterobacteriaceae, and thalline is direct rod shape, without pod membrane, active, amphimicrobian, has breathing and the metabolic way of two types of fermenting, at human airway, enteron aisle, be comparatively common in the foods such as animal intestinal and meat milk preparation.Hafnia alvei is a kind of conditioned pathogen, this bacterium can cause microbemia in the crowd of hypoimmunity, respiratory tract infection, urinary tract infections and other infection, it may have certain relation with gastroenteritis, in addition, muco-enteritis and the hepatosplenomegaly of laying hen can also be caused, the Animal diseases such as Epizootic septicemia in Bulgarian rainbow trout.

Typing of bacteria and authentication method mainly contain traditional phenotypic approach, serological method and molecular assay method.But along with molecular biological development, traditional serotype and authentication method also exist certain problem, diagnostic method as this in serotype needs a large amount of antiserum(antisera)s, and antiserum(antisera) general classes is incomplete, quantity not sufficient, a large amount of antiserum(antisera)s is in preparation and also there are some difficulties in storing.On the other hand serotype method length consuming time, sensitivity is low, loss is high, poor accuracy, often there is cross reaction between the antiserum(antisera) that different O antigen produces.Therefore, the serological diagnosis method set up based on Protocols in Molecular Biology becomes developing direction.

The surface antigen of hafnia alvei, existing O antigen also has H antigen, but mainly carries out somatotype with O antigen, and the research of O antigen is more thorough.Determine that the formal serotype scheme of hafnia alvei studies a laboratory proposition of the plum Qi Nikefu institute of vaccine and serum by Moscow, Russia at present, confirmed 39 O and 36 H type.This sorting technique is the prefered method of epidemiology relation between research bacterial strain always, and in comparison from particularly useful in the bacterial strain of the local different time of difference.Also be more and more subject to people's attention for its Molecular Identification, it is fast and be easy to the extensive advantage used that molecular Biological Detection technology has speed, is the developing direction of the pathogenic microbes detect of generally acknowledging in the world at present.

In recent years, increasing molecular engineering is used for the somatotype of pathogenic bacteria, qualification, detection and disease screening, comprises transcribed spacer (ITS) sequential analysis, random amplification length polymorphism (RAPD) is analyzed, rDNA restriction fragment length polymorphism (RFLP) is analyzed.Molecular biology method not only can be used for the rapid serum somatotype examination of hafnia alvei, and stable qualification result can make up the deficiency of phenotypic characteristic authentication method.Compare with traditional sensing techniques, these are based on the molecular detection technology of polymerase chain reaction (PCR), do not need the process such as separation, pure culture through pathogenic bacteria, and have the advantages such as quick, sensitive, high specificity.

Polymerase chain reaction technology (Polymerasechainreaction, be called for short round pcr) admitted at present as microorganism detection technology and promoted, this technology has relative to traditional method that high-throughput, detection speed are fast, high specificity, sensitivity advantages of higher, only need simply increase bacterium process to sample, again by the centrifugal and detailed bacterium DNA profiling of cracking, just can to increase target sequence in the PCR process under the mediation of high specific primer, reach the object detected in sample whether containing invasive organism to be measured.The amplification procedure of PCR only needs 1.5 hours.This greatly improves working speed beyond doubt to inspection and quarantine department and Clinical Laboratory, also reduces job costs simultaneously.

Summary of the invention

The object of the present invention is to provide and the present invention relates to hafnia alvei G5897, the Nucleotide that G5898, G5900 are special, it is characterized in that described Nucleotide has:

1) one in the Nucleotide shown in SEQIDNO:1-6;

2) with the one in the Nucleotide of the nucleotide complementary shown in SEQIDNO:1-6; Described SEQIDNO:1-6 is as follows:

Present invention also offers a kind of PCR kit, comprise PCR primer, dNTP, damping fluid and archaeal dna polymerase, described PCR primer is the one in the Nucleotide such as shown in SEQIDNO:1-6.Described test kit, also comprises following reagent: 10mMdNTP30 μ l; 10 × enzyme spcificity reaction buffer 50 μ l; 5U/ μ l hot resistant DNA polymerase 5 μ l; The upstream and downstream primer that each reference culture of mix primer 10 μ l(is special, odd number is upstream primer, and even numbers is downstream primer G5897 is SEQIDNO:1-2; G5898 is SEQIDNO:3-4; G5900 is SEQIDNO:5-6); Positive reference substance 10 μ l primer; Negative controls 10 μ l; ddH ₂o5ml.

Wherein said PCR primer is preferably the one in described Nucleotide as shown in SEQIDNO:1-6.

The present invention further discloses hafnia alvei G5897, the special SEQIDNO:1-6 Nucleotide of G5898, G5900 for the preparation of detection hafnia alvei G5897, the PCR kit of G5898, G5900.

Hafnia alvei of the present invention refers to and samples in the crude extract of clinical samples culture or the crude extracts of the pure growth of hafnia alvei such as septicemia, urinary tract infection, wound infections.Collecting hafnia alvei and extracting genome is adopt ordinary method to prepare.

For the PCR kit of hafnia alvei, whole detecting step comprises sample pretreatment-amplification-electrophoresis detection result.Primer and the reagent required for PCR reaction system add in amplification pipe in advance, and pretreated sample only need be added amplification pipe by user, and start amplified reaction, simple and quick completes testing.

The present invention also provides a kind of Liquid Detection chip, comprises liquid phase magnetic bead and is connected to the oligonucleotide probe on liquid phase magnetic bead, and the corresponding primer corresponding to the fragment of correspondent probe place; Wherein said Nucleotide is preferably the Nucleotide as shown in SEQIDNO:1-6.

The present invention also provides a kind of micro-array, and it comprises above-mentioned Nucleotide; Wherein said Nucleotide is preferably the Nucleotide as shown in SEQIDNO:1-6.

The present invention further discloses hafnia alvei G5897, the application of Nucleotide in the gene chip for the preparation of detection hafnia alvei PCR kit, detection hafnia alvei, in detection hafnia alvei microarray that G5898, G5900 are special.Described detection hafnia alvei refers to detection and causes bronchitis, pneumonia, urinary tract infections, the bacterium of wound infection and septicemia.

Disclosed by the invention to hafnia alvei G5897, compared with prior art, tool of the present invention has the following advantages the special Nucleotide of G5898, G5900:

(1) practical

A kind of PCR reaction system that the present invention sets up, can detect hafnia alvei, provides the special primer used by serotype detection, utilizes this PCR method can detect clinical samples.

(2) accuracy is high

The present invention passes through the PCR reaction to the special gene of each serotype of hafnia alvei, and each sample obtains the band of an entry, will obtain object fragment compared with known length, and just can obtain strain number corresponding to hafnia alvei.

(3) testing cost is relatively low

The fields such as Food Hygiene Surveillance, environmental monitoring, the quarantine of commodity supervision and inspection can be applied to, and provide technology mode for other different pathogenic microbes detects combine.

Accompanying drawing illustrates:

Fig. 1 represents that G5897 special primer of the present invention detects hafnia alvei and other reference cultures electrophoresis result figure, wzygene P1 and P2 object band are 287bp, and remaining bacterial strain is without any band, and concrete bacterial strain information is in table 2;

Fig. 2 represents the species specific qualification electrophoresis result figure of G5897 special primer of the present invention, and wherein have detected 6 strain vibrios and 1 strain salmonella, 1 strain intestinal bacteria, all without any band, concrete bacterial strain information is in table 2;

Fig. 3 represents G5898's of the present invention wzygene P3 and P4 primer detect hafnia alvei and other reference cultures electrophoresis result figure, and object band is 241bp, and concrete bacterial strain information is in table 2;

Fig. 4 represents wzy gene P3 and the species specific qualification electrophoresis result figure of P4 primer of this G5898, and wherein have detected 6 strain vibrios and 1 strain salmonella, 1 strain intestinal bacteria, all without any band, concrete bacterial strain information is in Table;

Fig. 5 represents this G5900's wzygene P5 and P6 primer detect hafnia alvei and other reference cultures electrophoresis result figure, and object band is 286bp, and remaining bacterial strain is without any band, and concrete bacterial strain information is in table 2;

Fig. 6 represents G5900's of the present invention wzygene P5 and the species specific qualification electrophoresis result figure of P6 primer, wherein have detected 6 strain vibrios and 1 strain salmonella, 1 strain intestinal bacteria, all without any band, concrete bacterial strain information is in table 2;

Fig. 7 represents and uses hafnia alvei G5897 respectively, the electrophoresis result figure of the corresponding serotype of G5898, G5900 specific primers amplify, and concrete bacterial strain information is in table 2;

Wherein: hafnia alvei G5897, G5898, G5900 represent corresponding serotype primer amplification result, and first swimming lane is 2000bpDNAmarker or 1kbPlusDL.

Embodiment

Below in conjunction with specific embodiment, set forth the present invention further.These embodiments should be understood only be not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition as people such as Sambrook, molecular cloning: the condition described in laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989).Wherein hafnia alvei derives from Warsaw, POL (PCM, PolishCollectionofMicroorganismsatIITDPAN, Wroclaw).

embodiment 1: genomic extraction

37 DEG C of nutrient broth mediums cultivate hafnia alvei, collect bacterium, extract genome concrete steps as follows:

With 500ul50mMTris-HCl (pH8.0) and 10ul0.4MEDTA re-suspended cell, 37 DEG C of incubations 20 minutes, the N,O-Diacetylmuramidase then adding 10ul10mg/ml continues insulation 20 minutes.Add the Proteinase K of 3ul20mg/ml, 15ul10%SDS afterwards, 50 DEG C of incubations 2 hours, then add the RNase of 3ul10mg/ml, 65 DEG C of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor, then use isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1) solution extracting twice, get supernatant liquor, then with isopyknic ether extraction to remove remaining phenol.Supernatant liquor 2 times of volume ethanol precipitation DNA, roll out DNA with glass yarn and wash DNA with 70% ethanol, being finally resuspended in 30ulTE by DNA.Genomic dna is detected by the agarose gel electrophoresis of 0.4%.

embodiment 2:sequence is decoded

Extract hafnia alvei G5897, G5898, the genome of G5900 reference culture, by Solexapair-end sequencing technologies, the sequence that genome sequencing obtains this O serotype is carried out to each standard gene group of hafnia alvei, Blast and PSI-Blast is used to carry out sequence alignment, TMHMMServer2.0program is adopted to carry out transmembrane structure prediction, ClustalWprogram is used to carry out sequence alignment and screen conservative and specific gene fragment, final O antigen gene cluster sequence and the decoding result obtaining each serotype of hafnia alvei.

embodiment 3: design of primers

Decode situation according to gene cluster and build storehouse comparison result, finding that wzy and wzx gene is the gene of hafnia alvei O antigen-specific, so choose this gene specific section design special primer.Because wzy is more special, thus main with wzy gene for target gene.

Design of primers is the core of this invention.Design primer designs according to the specific gene described in document.These two genes of wzx and wzy are genes more special in hafnia alvei O antigen gene cluster, can as the target gene of Serotype Identification.Said gene is imported PrimerPremier5 and carry out design of primers, the length of primer is preferably between 18 ~ 24bp, and Tm value is at 53 ~ 58 DEG C.Each gene design pair of primers, has single purpose band.

In Genbank, BLAST is carried out after design of primers, the primer of design can not be too high with the sequence similarity of other nearly edge bacterium, so just can guarantee that this primer only increases in the predetermined position of oneself, and not produce positive reaction with the nearly edge bacterium in the environment of other nearly edge bacterium or collect specimen.This point is very important for the success or failure of the generation and experiment of avoiding non-specific band.

The primer designed is as shown in table 1.

Table 1 is for the primer sequence of PCR

embodiment 4: the screening of special primer

Have collected hafnia alvei G5897, the reference culture of G5898, G5900 comprises: 6 strain Vibrios, the specificity of 1 strain Salmonella strain and 1 strain coli strain checking primer, and strain number and source are in table 2; There is provided simultaneously hafnia alvei enter and leave the border electronic edition prove, file papery of affixing one's seal version in Tianjin board of health, use when examining, see other supporting documentss:

Table 2 is for the bacterial strain of specific detection

The PCR system that gene identification primer screening is used be 5 μMs of primer 0.4 μ l, 10 × enzyme spcificity reaction buffer 2.5 μ l, 10mMdNTP0.25 μ l, 5U/ μ l hot resistant DNA polymerase 0.2 μ l and 3 μ l testing sample template in the thin-walled PCR pipe of 0.2ml, last ddH ₂o supplies 25 μ l.All primers all obtain positive findings in bacterial strain corresponding separately, do not obtain any PCR primer band in other groups.

Reaction cycle parameter in PCR instrument in this step comprises the sex change of DNA, renaturation, the temperature and time of extension, cycle index, is specially:

Early stage for enable sex change reach required temperature and required early stage treating processes a circulation be 95 DEG C, 5 minutes;

Denaturation temperature and time are 95 DEG C, 45 seconds;

Renaturation temperature and time is 53 DEG C/58 DEG C, 45 seconds;

Elongating temperature and time are 72 DEG C, 1 minute;

The cycle index of sex change, renaturation, extension is 35 circulations;

For stable amplified production and carry out one circulation temperature and time be 72 DEG C, 5 minutes

Wherein, G5897, G5898, G5900 use 53-6 DEG C of amplification, and above-mentioned steps is electrophoresis amplified production in electrophoresis equipment, and the concrete steps of record result are:

get 2 ~ 5 μ l amplified productions to mix with the volume ratio of 1:1 with 6 × tetrabromophenol sulfonphthalein sample-loading buffer;

mixed solution is splined on the sepharose of 1.0%;

by agarose gel electrophoresis 120v voltage stabilizing electrophoresis about 30 minutes, contrast with DL2000Marker;

observe and record result.

Substantially terminated by the work of primer screening after basic PC R reaction, necessary length adjustment is little on the impact of W-response condition, and the primer sequence used in the present invention is all summed up in Table 1.

Table 1 is for the primer sequence of PCR

the preparations and applicatio of embodiment 6:PCR detection kit

1, the composition of PCR kit:

dNTP(10mM)30μl；

10 × Buffer (10 × enzyme spcificity reaction buffer) 50 μ l;

Taq polysaccharase (5U/ μ l hot resistant DNA polymerase) 5 μ l;

PCR primer (5 μMs) SEQIDNO:1-610 μ l;

Positive reference substance (KP) 10 μ l;

Negative controls (KN) 10 μ l;

ddH ₂O5ml；

Each test kit can be used for detection 10 samples.

Wherein 10 × Buffer, dNTP, Taq polysaccharase are provided by precious biotechnology company limited; Primer is that the sequence of designed, designed is supplied to the synthesis of Shanghai Ying Jun biotech company; Positive reference substance, negative controls and ddH ₂o is prepared voluntarily by us.

2, plant and instrument

Wherein 10 × Buffer, dNTP, Taq polysaccharase are provided by the raw work in Shanghai; Primer is that the sequence of designed, designed is supplied to the synthesis of Shanghai Ying Jun biotech company; Positive reference substance, negative controls and ddH2O are prepared voluntarily by us.The equipment PCR instrument (having another name called DNA thermal cycling amplification instrument) of experiment, electrophoresis equipment (comprising electrophoresis apparatus and electrophoresis chamber), gel imaging instrument ,-20 DEG C of refrigerators, supercentrifuge, micropipet and 0.2mlPCR thin-walled tubes.

3, the use specific examples of PCR kit

The PCR detection method using above-mentioned PCR kit to detect hafnia alvei comprises the steps:

(1) environmental sample template to be measured is extracted;

(2) add in PCR thin-walled tube, dNTP, 10 × Buffer, Taq polysaccharase, primer, testing sample template and ddH ₂o mixes;

(3) mixture mixed in thin-walled PCR pipe is increased in PCR instrument;

(4) electrophoresis amplified production in electrophoresis equipment, record result;

(5) analyze and carry out result judgement.

Environmental sample template in above-mentioned steps (1) is sample the crude extract in clinical samples hafnia alvei cultures such as septicemia, urinary tract infection, wound infections, the crude extract of the pure growth of hafnia alvei or pure dna, or positive reference substance and negative controls.

The extracting method of the environmental sample template in above-mentioned steps (1) is:

get 1.5ml culture, under 12000rpm condition centrifugal 1 minute, remove supernatant liquor;

get the ddH of 500 μ l ₂the resuspended precipitation of O, under 8000rpm condition centrifugal 5 minutes, removes supernatant liquor, dries;

get 100 μ lddH ₂the resuspended precipitation of O, water-bath 10 minutes in 100 DEG C of boiling water;

be placed on ice after 10 minutes again, under 12000rpm condition centrifugal 2 minutes;

5. 3 μ l middle layer supernatant are got as pcr template

Reaction cycle parameter in PCR instrument in above-mentioned steps (3) comprises the sex change of DNA, renaturation, the temperature and time of extension, cycle index, is specially:

Early stage for enable sex change reach required temperature and required early stage treating processes a circulation be 95 DEG C, 5 minutes;

Denaturation temperature and time are 95 DEG C, 45 seconds;

Renaturation temperature and time is 53 DEG C/58 DEG C, 1 minute;

Elongating temperature and time are 72 DEG C, 1 minute;

The cycle index of sex change, renaturation, extension is 35 circulations;

For stable amplified production and carry out one circulation temperature and time be 72 DEG C, 5 minutes.

Above-mentioned steps (4) electrophoresis amplified production in electrophoresis equipment, the concrete steps of record result are:

get 2 ~ 5 μ l amplified productions to mix with the volume ratio of 5:1 with 6 × tetrabromophenol sulfonphthalein sample-loading buffer;

mixed solution is splined on the sepharose of 1.0%;

by agarose gel electrophoresis 120v voltage stabilizing electrophoresis about 30 minutes, contrast with DL2000Marker;

observe and record result.

The present invention by configuration a kind of detect hafnia alvei can industrialization produce PCR kit, PCR detection method is needed the combination of components of use together, during use, extract testing sample, just quick, sensitive, easy detection can be carried out through comparatively simple operation sequence simultaneously, in test kit, the consumption of each component and concentration are test gained, and detect with this test kit the testing installation that hafnia alvei uses simple, testing cost is low.

Use object that is positive and negative controls to be for the whole operating process of Quality Control, judge accurately to draw.If containing hafnia alvei object Strain type, then can observe the band with positive reference substance same position from electrophoresis result; If not containing hafnia alvei object Strain type, then the same with negative controls do not have this band.

The amount of reagent that one-time detection of the present invention is tested in the test kit used sees the following form shown in 3, and DNA profiling amount is 3 μ l

Table 3 one-time detection tests the amount of reagent in the test kit used

Hot resistant DNA polymerase in the present invention is Taq enzyme.

Above-mentioned positive reference substance is determined it is the sample of hafnia alvei reference culture, and negative controls is then for determining it is not the sample of hafnia alvei through laboratory.

This PCR kit bacteria suspension of hafnia alvei carries out pcr amplification, with consistent as template acquired results through extracting the DNA obtained.Susceptibility and specificity indifference, like this, can save the extraction step of template DNA, working method is simplified.Meanwhile, compare routine biochemistry detection method, the testing sample that present method adopts can be directly clinical sample nutrient solution, or to detection sample carry out simple separation cultivate just can detect, thus save manpower and materials.

4, the providing of testing sample

Have collected hafnia alvei G5897, G5898, G5900 reference culture, and 6 other bacterial strains of strain Vibrio, the specificity of 1 strain Salmonella strain and 1 strain coli strain checking primer, strain number and source are in table 2.

The above, only operation of the present invention and implementation method, not any pro forma restriction is done to the present invention, every above embodiment is done according to technical spirit of the present invention any simple modification, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.

SEQUENCELISTING

<110> Nankai University

<120> to hafnia alvei G5897, the Nucleotide that G5898, G5900 are special and application thereof

<160>6

<170>PatentInversion3.5

<210>1

<211>23

<212>DNA

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tgctatctagtgtgcttgttttg23

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<212>DNA

<213> artificial sequence

<400>2

cattgacggtgccaaatt18

<210>3

<211>22

<212>DNA

<213> artificial sequence

<400>3

attcaaattgcctcacattatt22

<210>4

<211>20

<212>DNA

<213> artificial sequence

<400>4

ccaactgtcgatagtgtgcc20

<210>5

<211>24

<212>DNA

<213> artificial sequence

<400>5

gtgttaaatcgatttaacgaatta24

<210>6

<211>24

<212>DNA

<213> artificial sequence

<400>6

acatcaaacgttaacacagagtaa24

Claims (6)

1. couple hafnia alvei G5897, the Nucleotide that G5898, G5900 are special, is characterized in that described Nucleotide has: the one in the Nucleotide shown in SEQIDNO:1-6.

2. according to claim 1 to hafnia alvei G5897, the Nucleotide that G5898, G5900 are special, wherein above-mentioned Nucleotide may be used for preparation detection hafnia alvei G5897, G5898, G5900 PCR kit, gene chip or microarray; Wherein said hafnia alvei refers to and samples in the crude extract of clinical samples culture or the crude extracts of the pure growth of hafnia alvei such as septicemia, urinary tract infection, wound infections.

3. one kind according to claim 1 to hafnia alvei G5897, G5898, the PCR kit of the Nucleotide that G5900 is special, comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase, and described PCR primer is the one in the Nucleotide such as shown in SEQIDNO:1-6; Also comprise following reagent: 10mMdNTP30 μ l; 10 × enzyme spcificity reaction buffer 50 μ l; 5U/ μ l hot resistant DNA polymerase 5 μ l; Primer 10 μ l; Positive reference substance 10 μ l; Negative controls 10 μ l; ddH ₂o1ml.

4. described in claim 1 to hafnia alvei G5897, the special Nucleotide of G5898, G5900 for the preparation of detecting hafnia alvei PCR kit, detect application in the gene chip of hafnia alvei.

5. described in claim 1 to hafnia alvei G5897, the special Nucleotide of G5898, G5900 is for the preparation of the application detected in hafnia alvei microarray.

6. the application described in claim 4-5, wherein said detection hafnia alvei refers to detection and causes bronchitis, pneumonia, urinary tract infections, the bacterium of wound infection and septicemia.