CN104694620A - LAMP (loop-mediated isothermal amplification) and primer set adopted molecular detection method of a variety of microorganisms - Google Patents
LAMP (loop-mediated isothermal amplification) and primer set adopted molecular detection method of a variety of microorganisms Download PDFInfo
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Abstract
The invention discloses an LAMP (loop-mediated isothermal amplification) and primer set adopted molecular detection method of a variety of microorganisms, and the molecular detection method can be applied to the fields of animal and plant quarantine, animal and plant protection, food controlling and monitoring, environment monitoring and protecting, health and disease prevention and clinical diagnosis.
Description
One. technical field
The invention belongs to field of molecular detection.
Two. background technology
The molecular detecting method detected based on nucleic acid (DNA/RNA) has started to carry out on a large scale entering in the past by additive method (traditional method or the method for antibody) prevailing test market with its high sensitivity and specificity more than ten years in the past.Molecular diagnosis method provides and is applicable to zoic universal method, and it can be used for carrying out responsive and special DNA/RNA detection to the biology being in life cycle different steps.Polymerase chain reaction (PCR), particularly PCR in real time (RT-PCR) are up to the present the most successful molecular detecting methods.But, the complicacy of RT-PCR detecting instrument and expensive price, detection speed slowly, and the Sample Preparation Procedure of complexity greatly limit its practical application.
Isothermal amplification, it does not need repeatedly temperature cycle, accelerates mensuration, and no longer needs complicated and expensive equipment.Ring mediated isothermal amplification method (Loop-mediated isothermal amplification, LAMP) be develop the most perfect isothermal nucleic acid amplification method up to now, it uses the multipair primer of particular design, form single stranded circle intermediate in the reaction, thus make DNA amplification reaction can in constant temp about 65 DEG C generation.The sensitivity that LAMP technology provides is similar with RT-PCR method with specificity levels, has exceeded Standard PCR.LAMP experimental procedure is simple, and speed of response is fast, to inhibitor better tolerance, sensitivity and specificity high, be the molecular detecting method of best overall equilbrium various aspects of performance.
Although LAMP technology has a lot of advantage, supporting universal testing kit also appears on the market, but the LAMP of each biology detects the primer needing special design, the detection of unknown species often needs to use carries out repeatedly LAMP reaction for multiple primer that is similar or close species, this considerably increases the complicacy of detection, also improve the cost of detection.
Three. summary of the invention
In order to overcome the deficiencies in the prior art, the invention provides a kind of LAMP reaction method detected for unknown species.By used in combination according to specific ratio for multiple LAMP primer that is similar or close species, the original so multiple LAMP carried out separately that will separate react the several reactions just merging into or a small amount of, thus the LAMP that enormously simplify unknown species detects, and reduces testing cost.
Four. accompanying drawing explanation
Fig. 1 uses the special primer group LAMP reaction detection capsicum trypetid DNA for capsicum trypetid
At this, use 4 melon trypetid DNA sample, 7 Bactrocera correcta DNA sample, 5 citrus fruit fly DNA sample and 2 capsicum trypetid DNA sample.The DNA cloning curve that A:RT-PCT instrument detects in real time, only has when using capsicum trypetid DNA sample and special DNA cloning just detected; B: the LAMP DNA amplification product using capsicum trypetid DNA sample to obtain shows close melting temp.
Fig. 2 uses the special primer group LAMP reaction detection melon trypetid for melon trypetid
At this, use 4 melon trypetid DNA sample, 7 Bactrocera correcta DNA sample and 5 citrus fruit fly DNA sample.The DNA cloning curve that A:RT-PCT instrument detects in real time, only has when using melon trypetid DNA sample and special DNA cloning just detected; B: the LAMP DNA amplification product using melon trypetid DNA sample to obtain shows close melting temp.
Fig. 3 uses the special primer group LAMP reaction detection Bactrocera correcta for Bactrocera correcta
At this, use 3 Bactrocera correcta DNA sample, 1 melon trypetid DNA sample, 1 capsicum trypetid DNA sample and 1 peach trypetid DNA sample.A:GenieII isothermal duplication fluorescence detector detects other DNA cloning curve in real time, only has when using Bactrocera correcta DNA sample and special DNA cloning just detected; B: the LAMP DNA amplification product using Bactrocera correcta DNA sample to obtain shows close melting temp.
Fig. 4 uses the special primer group LAMP reaction detection peach trypetid for peach trypetid
At this, use 3 Bactrocera correcta DNA sample and 3 peach trypetid DNA sample, other two reactions do not add DNA as negative control but add distilled water.The DNA cloning curve that A:Genie II isothermal duplication fluorescence detector detects in real time, only has when using peach trypetid DNA sample and special DNA cloning just detected; B: the LAMP DNA amplification product using Bactrocera correcta DNA sample to obtain shows close melting temp.
Fig. 5 uses the LAMP detection reaction for the mix primer of multiple trypetid
At this, use 2 melon trypetid DNA sample, 2 Bactrocera correcta DNA sample, 2 peach trypetid DNA sample and 2 capsicum trypetid DNA sample.The DNA cloning curve that A:GenieII isothermal duplication fluorescence detector detects in real time, uses the LAMP reaction for the mix primer of multiple trypetid can identify melon trypetid, Bactrocera correcta, peach trypetid and capsicum trypetid DNA sample; B: the DNA amplification product that identical type DNA sample obtains shows close melting temp.
Five. embodiment
The following examples can help the present invention of those skilled in the art's comprehend, but do not limit the present invention in any way.The trypetid DNA sample that the present embodiment uses, extracts: capsicum trypetid (Bactrocera latifrons), melon trypetid (Bactrocera cucurbitae), Bactrocera correcta (Bactrocera correcta), peach trypetid (Bactrocera zonata) and citrus fruit fly (Bactrocera dorsalis) from following trypetid.All trypetid DNA sample are all extract from the trypetid sample of censorship in plant production Science Institute of agrotechnique research department of Swiss Confederation, and all trypetid samples are all identified through DNA sequencing.
1. for the primer of LAMP detection:
(1) for the special primer group of capsicum trypetid
F3-lat：5’-CTATTTTCTCATTACACTTAGCCGGA-3’
B3-lat：5’-CTCCAGCGGGGTCAAAGAAT-3’
FIP-lat：5’-TCGGTCGAATGAAATTCCTGTTGATCCTCAATCTTAGGAGCAGTTAAC-3’
BIP-lat：5’-GCCTCTTTTCGTTTGAGCAGTTGTCCYGCTAAAACTGGTAGTGAC-3’
LoopF-lat：5’-CGTATGTTGATTACTGTTGTGATG-3’
LoopB-lat：5’-TACTAACGGCYCTATTACTCTTAC-3’
(2) for the special primer group of melon trypetid
F3-cuc：5’-GGAGCTGGTACAGGTTGAACT-3’
B3-cuc：5’-GATAGAAGTAAAAGRAGAGCTGTC-3’
FIP-cuc：5’-CCTAAAATTGATGAAATACCAGCTAAATGCTTTCATCAATTATCGCTCATGGT-3’
BIP-cuc：5’-GCCGTAAATTTCATTACTACAGTRATTAATATCRGCTCAAACGAATAAAGGTATC-3'
LoopF-cuc：5'-AGAGAAAAAATAGCTAAATCAACTGAG-3’
LoopB-cuc：5’-GCGATCAACAGGAATCACATTTGAC-3’
(3) for the special primer group of Bactrocera correcta
F3-cor：5’-CTGGTACAGGTTGAACAGTTTACCCT-3’
B3-cor：5’-CTGGTAGTGATAATAGAAGCAATAAA-3’
FIP-cor：5’-AAATTGAGGAAATACCAGCTAAGTG-CCCCTATCATCTGTTATTGCTCAC-3’
BIP-cor：5’-AGGAGCAGTAAATTTTATCACAACCGTGCTGTTAATACAACTGCTCAAACGAAT-3’
loopF-cor：5’-GAGTGAGAAAATAGCTAGATCAACC-3’
loopB-cor：5’-GCGATCGACAGGAATTTCATTTGAC-3’
(4) for the special primer group of peach trypetid
F3-zon：5’-CTGGTACAGGTTGAACAGTTTAT-3’
B3-zon：5’-CTGGYAATGATAATAGAAGTAATAGG-3’
FIP-zon：5’-GAATTGAGGAAATACCRGCTAAGTCCCTATCATCTGTTATTGCTCAC-3’
BIP-zon：5’-GGAGCAGTTAATTTTATYACAACTGTTATGCTGTTAATACAACTGCTCAAACGAAG-3’
LoopF-zon：5’-GAAGTGAGAAAATAGCTAGATCAACTGAA-3’
LoopB-zon：5’-ACGTTCAACAGGAATTTCATTTGAT-3’
(5) for capsicum trypetid, melon trypetid, Bactrocera correcta and peach trypetid mix primer
All primers by above described in (1)-(4) mix use.
2.LAMP reaction system:
Following reagent is added in each LAMP reaction tube (Optigene):
DNA extraction liquid 1 μ l
2x reaction solution (Isothermal Master Mix, Optigene)
F3 and B3 primer final concentration is respectively 0.2 μM
F1P and B1P primer final concentration is respectively 2 μMs
LoopF and LoopB primer final concentration is respectively 1 μM;
Will for capsicum trypetid, melon trypetid, when the mix primer of Bactrocera correcta and peach trypetid carries out LAMP reaction, the final concentration of often kind of primer is also described above, and total reaction volume being mended with aquae destillata is 15 μ l
Reaction, in ABI7500RT-PCR instrument or Genie II isothermal duplication fluorescence detecting system, 65 DEG C, carries out 60 minutes.Containing DNA fluorescence dye SYBR Green I in reaction solution (Isothermal Master Mix), thus can carry out Real-Time Monitoring to DNA cloning, and carry out the analysis of DNA melting point to determine whether specific amplification.
3.LAMP reaction detection trypetid result
(1) the special primer group LAMP reaction detection capsicum trypetid for capsicum trypetid is used
Add the special primer group for capsicum trypetid in the reaction, react and carry out in ABI7300RT-PCR instrument.Use the LAMP reaction for the special primer group of capsicum trypetid can only identify capsicum trypetid DNA (Figure 1A) specifically, the D N A that the melting point analysis confirmation of amplification D N A amplifies has similar melting point, is that special D N A increases.
(2) the special primer group LAMP reaction detection melon trypetid for melon trypetid is used
Add the special primer group for melon trypetid in the reaction, react and carry out in ABI7300RT-PCR instrument.Use the LAMP reaction for the special primer group of melon trypetid can only identify melon trypetid DNA (Fig. 2 A) specifically, the all D N A amplified of melting point analysis confirmation of amplification D N A have similar melting point (Fig. 2 B), are that special D N A increases.
(3) the special primer group LAMP reaction detection Bactrocera correcta for Bactrocera correcta is used
Add the special primer group for Bactrocera correcta in the reaction, react and carry out in Genie II isothermal duplication fluorescence detector.Use the LAMP reaction for the special primer group of Bactrocera correcta can only identify Bactrocera correcta DNA specifically, and to capsicum trypetid, melon trypetid and peach trypetid D N A sample then Fails To Respond (Fig. 3 A), the all D N A amplified of melting point analysis confirmation of amplification D N A have similar melting point (Fig. 3 B), are that special D N A increases.
(4) the special primer group LAMP reaction detection peach trypetid for peach trypetid is used
Add the special primer group for peach trypetid in the reaction, react and carry out in Genie II isothermal duplication fluorescence detector.Use the LAMP reaction for the special primer group of peach trypetid can only identify peach trypetid DNA sample specifically, to Bactrocera correcta D N A sample then Fails To Respond (Fig. 4 A), the all D N A amplified of melting point analysis confirmation of amplification D N A have similar melting point (Fig. 4 B), are that special D N A increases.
(5) the LAMP detection reaction for the mix primer of multiple trypetid is used
Add the mix primer for multiple trypetid in the reaction, react and carry out in Genie II isothermal duplication fluorescence detector.Use the LAMP reaction for the mix primer of multiple trypetid can identify capsicum trypetid, peach trypetid, melon trypetid and Bactrocera correcta DNA sample (Fig. 5 A), the D N A that the DNA profiling of the melting point analysis confirmation trypetid mutually of the same race of amplification D N A amplifies has similar melting point (Fig. 5 B), is that special D N A increases.
Claims (2)
1. by ring mediated isothermal amplification LAMP reaction method used in combination for the LAMP primer group for different plant species.
2. according to claim 1, in the Animal or Plant Quarantine, fauna and flora protection, food control and monitoring, environmental monitoring and protection, health and epidemic prevention and clinical diagnosis field, for the primer mixture reagent for multiple biology or the test kit of LAMP reaction.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107674921A (en) * | 2017-11-16 | 2018-02-09 | 凭祥出入境检验检疫局综合技术服务中心 | A kind of quick visualization melonfly molecular detecting method |
| CN113817848A (en) * | 2021-10-28 | 2021-12-21 | 青岛农业大学 | Method for detecting Bactrocera minax (Royle) comatus based on visual LAMP technology and application |
| CN114058709A (en) * | 2020-07-29 | 2022-02-18 | 中国农业大学 | LAMP technology-based method for identifying Drosophila FARQ complex and special primer group thereof |
| CN114058707A (en) * | 2020-07-29 | 2022-02-18 | 中国农业大学 | LAMP kit for identifying Bactrocera mediterranei and application thereof |
| CN114058708A (en) * | 2020-07-29 | 2022-02-18 | 中国农业大学 | LAMP primer group for identifying African mango fruit flies and identification method thereof |
| CN114717324A (en) * | 2021-01-05 | 2022-07-08 | 中国农业大学 | LAMP primer group for identifying fresh cherry common flies and application thereof |
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| CN102329882A (en) * | 2011-10-15 | 2012-01-25 | 浙江省质量技术监督检测研究院 | Multiple rapid detection method, detection primer group and kit for three food-borne pathogenic bacteria |
| CN102586439A (en) * | 2012-02-28 | 2012-07-18 | 盛司潼 | Method for simultaneously and quickly detecting multiple nucleic acids |
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Patent Citations (3)
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| CN102021246A (en) * | 2010-12-02 | 2011-04-20 | 华南农业大学 | LAMP (loop-mediated isothermal amplification) detection method for rapidly identifying and detecting fasciola hepatica and fasciola gigantica and reagent box |
| CN102329882A (en) * | 2011-10-15 | 2012-01-25 | 浙江省质量技术监督检测研究院 | Multiple rapid detection method, detection primer group and kit for three food-borne pathogenic bacteria |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107674921A (en) * | 2017-11-16 | 2018-02-09 | 凭祥出入境检验检疫局综合技术服务中心 | A kind of quick visualization melonfly molecular detecting method |
| CN114058709A (en) * | 2020-07-29 | 2022-02-18 | 中国农业大学 | LAMP technology-based method for identifying Drosophila FARQ complex and special primer group thereof |
| CN114058707A (en) * | 2020-07-29 | 2022-02-18 | 中国农业大学 | LAMP kit for identifying Bactrocera mediterranei and application thereof |
| CN114058708A (en) * | 2020-07-29 | 2022-02-18 | 中国农业大学 | LAMP primer group for identifying African mango fruit flies and identification method thereof |
| CN114058709B (en) * | 2020-07-29 | 2023-08-01 | 中国农业大学 | LAMP technology-based identification method of Bactrocera farQ complex and special primer group thereof |
| CN114717324A (en) * | 2021-01-05 | 2022-07-08 | 中国农业大学 | LAMP primer group for identifying fresh cherry common flies and application thereof |
| CN113817848A (en) * | 2021-10-28 | 2021-12-21 | 青岛农业大学 | Method for detecting Bactrocera minax (Royle) comatus based on visual LAMP technology and application |
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Application publication date: 20150610 |