CN102010910A - Loop-mediated isothermal amplification technology-based plasmodium genus and species nucleic acid screening method - Google Patents
Loop-mediated isothermal amplification technology-based plasmodium genus and species nucleic acid screening method Download PDFInfo
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Abstract
The invention discloses a set of loop-mediated isothermal amplification technology-based plasmodium genus and species nucleic acid screening method and belongs to the field of biological detection. The method is performed by loop-mediated isothermal amplification (LAMP) technology, specific positions of target genes are amplified by using an LAMP technology platform through specific primers of plasmodium genera and specificity primers of plasmodium species, the plasmodium genera are screened or detected at a molecular level under assistance of positive and negative quality control and an internal control detection system, and plasmodium species screening or detection is performed on four kinds of plasmodia, namely Plasmodiumfalciparum, Plasmodiummalariae, Plasmodiumovale and Plasmodiumvivax which can make humans infected with malaria in plasmodium genus organisms. The invention has the characteristics that: the method is simple, economic and rapid, and has high sensitivity, high specificity and wide application prospect.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to a kind of based on the plasmodium of ring mediated isothermal amplification (LAMP) technology and the nucleic acid screening method of kind; Contain plasmodium falciparum (
Plasmodium falciparum), malariae (
Plasmodium malariae), Plasmodium ovale (
Plasmodium ovale) and Plasmodium vivax (
Plasmodium vivax)) nucleic acid screening method.
Background technology
The detection method of plasmodium and kind mainly is that microorganism culturing is identified and the multiplex PCR detection technique at present.(1) sediments microscope inspection, thick blood film, thin smear film sediments microscope inspection are still the gold standard that plasmodium detects.But there are some problems again in sediments microscope inspection as a kind of detection method.Because the plasmodium body is little, plesiomorphism is difficult to dye and detect, and experienced personnel just can accomplish correct diagnosis; Simultaneously, microscopy is wasted time and energy, and the detection that is not suitable for extensive sample is unfavorable for above-mentioned plasmodial general examination.(2) polymerase chain reaction (PCR), specificity is relatively poor, and advantage is that susceptibility can reach 98%～100%., but it needs relevant valuable equipments such as pcr amplification instrument, gel electrophoresis, is unfavorable for primary care epidemic prevention organization and fast-field evaluation.
At the beginning of 21 century, Notomi T etc. has developed a kind of constant temperature nucleic acid amplification theory of novelty, be so-called loop-mediated isothermal amplification method (Loop-Mediated Isothermal Amplification, LAMP), be characterized in 6/4 kinds of special primers of 8/6 zone design at target gene, utilize a kind of strand displacement Bst DNA polysaccharase in constant temperature (about 60 ℃) insulation dozens of minutes, can finish nucleic acid amplification reaction, the short period of time amplification efficiency can reach 10
9– 10
10Individual copy.Directly judge whether to react by amplified production electrophoresis or color reaction.This method does not need processes such as the thermally denature of template, long-time temperature cycle, loaded down with trivial details electrophoresis, ultraviolet visualization, the LAMP method has high specific, high efficiency, quick, cheap (do not need specific valuable test set, only need the thermostat container of simple structure), the easy characteristics such as (directly estimating judgement) that detect.
Summary of the invention
The purpose of this invention is to provide and a kind ofly be applied to plasmodium and kind, contain plasmodium falciparum (
Plasmodium falciparum), malariae (
Plasmodium malariae), Plasmodium ovale (
Plasmodium ovale) and Plasmodium vivax (
Plasmodium vivax)) nucleic acid detection method, detectivity is further improved, the 100-200 that the detectability of LAMP detection technique is higher than the conventional sense method has doubly so just effectively overcome the problem of the traditional microscope lens detecting method recall rate end of than; The 3rd, because classical polymerase chain reaction (PCR) amplification technique is had relatively high expectations (as the pcr amplification instrument to plant and instrument, agargel electrophoresis systems etc.), technology is comparatively complicated, be unfavorable for its widespread usage in primary care epidemic prevention, inspection and quarantine mechanism, and the LAMP detection technique has the plant and instrument requirement and detects (thermostat container that only needs simple structure), detection time short (0.5-1.0 hour), specificity height (at 6/4 kind of special primer of 8/6 zone design of target gene,) etc. characteristics, can use the gene amplification technology to be extensive use of in feeler mechanism of basic unit.
The present invention realizes with following technical scheme: a kind ofly be applied to examination plasmodium and kind, contain plasmodium falciparum (
Plasmodium falciparum), malariae (
Plasmodium malariae), Plasmodium ovale (
Plasmodium ovale) and Plasmodium vivax (
Plasmodium vivax)) detection method of nucleic acid, utilize ring mediated isothermal amplification (LAMP) technology to carry out, utilize the LAMP technology platform by using the specific region of primer amplified plasmodium and kind target gene, positive and negative Quality Control and internal reference detection architecture auxiliary time, from molecular level to plasmodium and kind (contain plasmodium falciparum (
Plasmodium falciparum), malariae (
Plasmodium malariae), Plasmodium ovale (
Plasmodium ovale) and Plasmodium vivax (
Plasmodium vivax)) nucleic acid screening or detection.
Further, in the examination detection method of plasmodium of the present invention and kind nucleic acid, in LAMP examination process, introduced yin and yang attribute control test system.The positive control quality control product is for being same as the amplification plasmodium or planting the particular target gene fragment, the negative control quality control product is inequality in amplification plasmodium or kind particular target gene fragment, and uses electrophoretic analysis or SYBR Green I fluorescence dye development process to detect internal reference and plasmodium and kind target gene amplified production simultaneously.
Further, in streptococcus aureus of the present invention, Salmonellas, Shigellae and Listeria monocytogenes nucleic acid detection method, it is characterized in that used LAMP reaction system is a series of reacted constituent compositions by optimizing, and has specific response procedures.
The present invention has strict discriminating sieving and diagnosis system, to guarantee the accuracy of detected result.This differentiates the sieving and diagnosis system by positive and negative quality control product, and internal reference quality control product and relevant amplification and detection architecture are formed.
Whether effectively the positive and the negative quality control product of differentiating the sieving and diagnosis system among the present invention are every batch of laboratory test results important symbols.For every batch of experiment, must introduce the detection of a negative quality control product, to guarantee that reaction system false positive results can not occur; For every batch of experiment, must introduce the detection of a plasmodium and kind positive quality control product, to guarantee that reaction system false negative reaction can not occur.At the positive quality control product of plasmodium and kind is the plasmid of artificial constructed above-mentioned plasmodium target sequence to be checked.
Specific amplification products in the differential diagnosis of the present invention system designs by the contriver, prepares by the nucleic acid synthesis device.The nucleic acid synthesis device can be selected different manufacturers for use, and the product of different model also can entrust relevant genome company synthetic.All primers are according to LAMP design of primers principle, utilize ICBgyrB database and RIDOM database seek plasmodium and kind (contain plasmodium falciparum (
Plasmodium falciparum), malariae (
Plasmodium malariae), Plasmodium ovale (
Plasmodium ovale) and Plasmodium vivax (
Plasmodium vivax)) the LAMP gene order, utilize software to choose its specific sequence, utilize PrimerExplorer IV software further to analyze, the design 5 the cover primers, respectively at above-mentioned plasmodium and kind.LAMP system and response procedures in the differential diagnosis of the present invention system design by the contriver.The nucleic acid specificity primer sequence of Proteromonas and kind, LAMP reaction system and response procedures are as follows:
One, loop-mediated isothermal amplification (LAMP) primer
The LAMP reaction primer sequence of table one plasmodium is formed
| Primer | Classification | Sequence is formed |
| F3 | Preceding outer primer | 5’- GTATCAATCGAGTTTCTGACC?-3’ |
| B3 | Back outer primer | 5’- CTTGTCACTACCTCTCTTCT?-3’ |
| FIP | Preceding inner primer | 5’- TCGAACTCTAATTCCCCGTTACCTATCAGCTTTTGATGTTAGGGT?-3’ |
| BIP | Back inner primer | 5’- CGGAGAGGGAGCCTGAGAAATAGAATTGGGTAATTTACGCG?-3’ |
Table two plasmodium falciparum (
Plasmodium falciparum) LAMP reaction primer sequence form
| Primer | Classification | Sequence is formed |
| F3 | Preceding outer primer | 5’- TGTAATTGGAATGATAGGAATTTA?-3’ |
| B3 | Back outer primer | 5’- GAAAACCTTATTTTGAACAAAGC?-3’ |
| FIP | Preceding inner primer | 5’- AGCTGGAATTACCGCGGCTG?GGTTCCTAGAGAAACAATTGG?-3’ |
| BIP | Back inner primer | 5’- TGTTGCAGTTAAAACGTTCGTAGCCCAAACCAGTTTAAATGAAAC?-3’ |
Table three malariae (
Plasmodium malariae) LAMP reaction primer sequence form
| Primer | Classification | Sequence is formed |
| F3 | Preceding outer primer | 5’- CAAGGCCAAATTTTGGTT?-3’ |
| B3 | Back outer primer | 5’-?CGGTTATTCTTAACGTACA -3’ |
| FIP | Preceding inner primer | 5’- TATTGGAGCTGGAATTACCGCGATGATGGGAATTTAAAACCT?-3’ |
| BIP | Back inner primer | 5’- AATTGTTGCAGTTAAAACGCCTATGTTATAAATATACAAAGCATT?-3’ |
Table four Plasmodium ovale (
Plasmodium ovale) LAMP reaction primer sequence form
| Primer | Classification | Sequence is formed |
| F3 | Preceding outer primer | 5’- GGAATGATGGGAATTTAAAACC?-3’ |
| B3 | Back outer primer | 5’- GAATGCAAAGAACAGATACGT?-3’ |
| FIP | Preceding inner primer | 5’- TATTGGAGCTGGAATTACCGCGTTCCCAAAATTCAATTGGAGG?-3’ |
| BIP | Back inner primer | 5’- GTTGCAGTTAAAACGCTCGTAGTGTATTGTCTAAGCATCTTATAGCA?-3’ |
Table five Plasmodium vivax (
Plasmodium vivax) LAMP reaction primer sequence form
| Primer | Classification | Sequence is formed |
| F3 | Preceding outer primer | 5’- GGAATGATGGGAATTTAAAACCT?-3’ |
| B3 | Back outer primer | 5’-?ACGAAGTATCAGTTATGTGGAT -3’ |
| FIP | Preceding inner primer | 5’- CTATTGGAGCTGGAATTACCGCTCCCAAAACTCAATTGGAGG?-3’ |
| BIP | Back inner primer | 5’- AATTGTTGCAGTTAAAACGCTCGTAAGCTAGAAGCGTTGCT?-3’ |
Two, plasmodium that the present invention includes and kind (contain plasmodium falciparum (
Plasmodium falciparum), malariae (
Plasmodium malariae), Plasmodium ovale (
Plasmodium ovale) and Plasmodium vivax (
Plasmodium vivax)) the LAMP reaction system composed as follows:
Table six plasmodium and kind LAMP reaction system are formed
| Sequence number | Component | Dosage (μ l) |
| 1 | 2×U-LAMP Mix 1 | 10ul |
| 2 | 10mmol/L primer Mix 2 | 1μl |
| 3 | Template DNA | 2μl |
| 4 | 8000U/ml Bst enzyme | 0.8μl |
| 5 | Distilled water | 6.2μl |
| 6 | Add up to | 20 |
Remarks: 1,2 * U-LAMP Mix is composed as follows: 10 * Bst DNA Polymerase Buffer, 10 μ L;
10mmol/L?dNTP,16?μL;?150?mmol/L?MgSO
4,6?ul;?4mol/L?Betaine?,16?μL;
Supply water to 50 μ L.
2, primer Mix is composed as follows: each primer of 50mmol/L mixes, and supplies water to 200ul, wherein: preceding outer primer F3,8 ul; Back outer primer B3,8 ul; Preceding inner primer FIP, 32 ul; Back inner primer BIP, 32 ul.
Three, amplification program: amplification program is: above-mentioned reaction system places the PCR instrument, 59.6 ℃ of 1 hour → 80 ℃ 10 minutes → 10 ℃ preservations.
Four, reaction end detection method: after reaction finishes, add 1 μ L SYBR Green I fluorescence dye, whether mixing, observing response pipe color change or get the above-mentioned plasmodium LAMP of 5 μ L reaction product, carry out electrophoresis detection on 2% sepharose.Whether observe has the scalariform band to produce.
The detection method of differential diagnosis of the present invention system, undertaken by following step successively:
(1) nucleic acid extraction: get 200 μ l sample blood samples in the lml centrifuge tube, destroy cytolemma, abandon supernatant after 7000g is centrifugal, add people's DNA extraction liquid (10mmol/L Tris-HCl, pH8.0 with the saponin of final concentration 0.2%; 5mmol/L EDTA, pH 8.0; LOOmmol/L NaCI; 0.5% SDS; 0.2mg/ml digesting protein Proteinase K) adds final concentration 0.3mol/L sodium-acetate and two volumes ice ethanol sedimentation in the saturated phenol of equal-volume/chloroform extracting Deproteinization, supernatant
DNA, the 8O% washing with alcohol behind the dry air DNA, adds the template DNA of 30 μ l sterilization distilled water as amplification.
(2) LAMP reaction: prepare above-mentioned reaction system, with the reaction system mixing, divide and install in the PCR reaction tubes of 0.2ml, every pipe 18 μ l, add the extractive template of (1) step, every pipe 2ul, positive reference substance and each 2ul of negative control product in the PCR reaction tubes respectively, make positive control, negative control respectively, mix.Put into constant water bath box, set reaction conditions: 59.6 ℃, 60 minutes, 80 ℃ of 10 minutes termination reactions, 10 ℃ of preservations, reaction finish the back and add 1 μ l SYBR Green I dyestuff, mixing, observing response pipe whether color changes or gets the above-mentioned plasmodium LAMP of 5 μ L reaction product, carries out electrophoresis detection on 2% sepharose.。
(3) result judges: the reaction tubes of positive for bacteria genomic dna template can be observed color and becomes green, and negative control then is orange.After agarose gel electrophoresis detected, the reaction product of positive for bacteria genomic dna template had specific scalariform band to occur, and negative control does not then have band and occurs.
The invention has the beneficial effects as follows: method is easy, economical, quick, sensitive.
Description of drawings
Fig. 1 be plasmodium falciparum (
Plasmodium falciparum) LAMP reaction product dyeing figure.
Among the figure: 1, plasmodium falciparum genomic dna; 2, negative control; 3, blank.
Fig. 2 be plasmodium falciparum (
Plasmodium falciparum) LAMP amplified production gel electrophoresis figure.
Among the figure: M:DL2000; 1, plasmodium falciparum genomic dna; 2, negative control.
Embodiment
Embodiment
(1) nucleic acid extraction: from 10 hours bacteria suspension of 37 ℃ of shaking tables cultivations, draw 1ml bacterium liquid, the centrifugal 5min of 10000rpm, abandon supernatant, add the concussion of 100 μ l sterilized waters and shake up (or a picking 2-3 bacterium colony is in 100 μ l sterilized waters on the flat board of preserving) from 4 ℃ of refrigerators.100 ℃ are boiled 10min, and with centrifugal 5 min of 10000rpm, supernatant liquor is a genomic dna with desk centrifuge, can be directly as the template of nucleic acid amplification reaction.
(2) LAMP reaction: prepare above-mentioned reaction system, with the reaction system mixing, divide and install in the PCR reaction tubes of 0.2ml, every pipe 19ul, add the extractive template of (1) step, every pipe 1ul, positive reference substance and each 1ul of negative control product in the PCR reaction tubes respectively, make positive control, negative control respectively, mix.Put into constant water bath box, set reaction conditions: 65 ℃, 60 minutes, 80 ℃ of 10 minutes termination reactions, 10 ℃ of preservations, reaction finish the back and add 1 μ L Pico Green dyestuff, mixing, observing response pipe whether color changes or gets the above-mentioned bacterium LAMP of 5 μ L reaction product, carries out electrophoresis detection on 2% sepharose.
(3) result judges: the reaction tubes of positive for bacteria genomic dna template can be observed color and becomes green, and negative control then is colourless.After agarose gel electrophoresis detected, the reaction product of positive for bacteria genomic dna template had specific scalariform band to occur, and negative control does not then have band and occurs.
It is shown in Figure 2 that it the results are shown in accompanying drawing 1-.
Claims (4)
1. one kind based on the plasmodium of loop-mediated isothermal amplification technique and the nucleic acid screening method of kind, utilize ring mediated isothermal amplification (LAMP) technology to carry out, it is characterized in that, by using plasmodium Auele Specific Primer and species-specific primer, utilize the specific region of LAMP technology platform amplified target gene, assisting down of positive and negative Quality Control and internal reference detection architecture, from molecular level plasmodium is carried out examination or detection, and on this basis, to making four kinds of plasmodiums of human infection's malaria in this genus biology, comprise plasmodium falciparum (
Plasmodium falciparum), malariae (
Plasmodium malariae), Plasmodium ovale (
Plasmodium ovale) and Plasmodium vivax (
Plasmodium vivax) carry out examination of plasmodium kind or detection; The LAMP detection specificity primer of described plasmodium is:
The LAMP reaction primer sequence of plasmodium is formed
Described plasmodium falciparum (
Plasmodium falciparum) detect primer and be
Plasmodium falciparum (
Plasmodium falciparum) LAMP reaction primer sequence form
Described malariae (
Plasmodium malariae) LAMP detects primer and be
Malariae (
Plasmodium malariae) LAMP reaction primer sequence form
Plasmodium ovale (
Plasmodium ovale) LAMP detects primer and be
Plasmodium ovale (
Plasmodium ovale) LAMP reaction primer sequence form
Plasmodium vivax (
Plasmodium vivax) LAMP detects primer and be
Plasmodium vivax (
Plasmodium vivax) LAMP reaction primer sequence form
。
2. according to claim 1 based on the plasmodium of loop-mediated isothermal amplification technique and the nucleic acid screening method of kind, it is characterized in that utilizing the LAMP technology particular target nucleotide sequence zone of above-mentioned plasmodium and kind of increasing.
3. according to claim 1 based on the plasmodium of loop-mediated isothermal amplification technique and the nucleic acid screening method of kind, it is characterized in that in the described yin and yang attribute control test system that the positive control quality control product is for being same as the amplification plasmodium or planting the particular target gene fragment; The negative control quality control product is inequality in amplification plasmodium or kind particular target gene fragment, and use electrophoretic analysis, SYBR Green I fluorescence dye development process to detect negative and positive contrast and plasmodium and kind simultaneously, contain plasmodium falciparum, malariae, Plasmodium ovale and Plasmodium vivax particular target gene amplification product.
4. according to claim 1ly it is characterized in that used LAMP reaction system is a series ofly to form by the reacted constituents of optimizing, and has specific amplification program based on the plasmodium of loop-mediated isothermal amplification technique and the nucleic acid screening method of kind; Its LAMP reaction system is composed as follows:
Reaction system:
Wherein: 2 * U-LAMP Mix forms is: 10 * Bst DNA Polymerase Buffer, 10 μ L;
10mmol/L?dNTP,16?μL;?150?mmol/L?MgSO
4,6?ul;?4mol/L?Betaine?,16?μL;
Supply water to 50 μ L;
Primer Mix forms: each primer of 50mmol/L mixes, and supplies water to 200ul, wherein: preceding outer primer F3,8 ul; Back outer primer B3,8 ul; Preceding inner primer FIP, 32 ul; Back inner primer BIP, 32 ul;
Amplification program is: above-mentioned reaction system places the PCR instrument, 59.6 ℃ of 1 hour → 80 ℃ 10 minutes → 10 ℃ preservations;
Specific amplification program is: above-mentioned reaction system places the PCR instrument, 59.6 ℃ of 1 hour → 80 ℃ 10 minutes → 10 ℃ preservations.
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| CN102676651A (en) * | 2012-03-09 | 2012-09-19 | 广西壮族自治区疾病预防控制中心 | Quickly-nested PCR (polymerase chain reaction) malaria molecular diagnosis reagent kit and detection method |
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| CN106801097A (en) * | 2017-02-17 | 2017-06-06 | 湖北医药学院 | Ring mediated isothermal amplification method based on the exclusive gene of plasmodium falciparum ring bodies |
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| CN110331221B (en) * | 2019-08-19 | 2023-05-02 | 昆明医科大学 | Plasmodium gene diagnosis primer |
| CN113755621A (en) * | 2021-09-02 | 2021-12-07 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Reagent and kit for detecting plasmodium and distinguishing four plasmodium species by isothermal amplification and application of reagent and kit |
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Application publication date: 20110413 |