CN115386646A - Mycoplasma LAMP fluorescence detection primer composition and LAMP fluorescence detection method thereof - Google Patents
Mycoplasma LAMP fluorescence detection primer composition and LAMP fluorescence detection method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of mycoplasma LAMP detection, in particular to a mycoplasma LAMP fluorescence detection primer composition and an LAMP fluorescence detection method thereof, wherein the mycoplasma LAMP fluorescence detection primer composition consists of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3 and probes LF, LF1 and LF-Q; multiple LAMP reactions can be carried out; fluorescence and colloidal gold can be detected simultaneously, so that the cost and the time are saved; based on fluorescence detection, sensitivity is improved.
Description
Technical Field
The invention relates to the technical field of mycoplasma LAMP (loop-mediated isothermal amplification) detection, in particular to a mycoplasma LAMP fluorescent detection primer composition and an LAMP fluorescent detection method thereof.
Background
Since the invention of PCR technology in 1980, the nucleic acid detection based on PCR technology is rapidly applied to detection of clinic, food and environment, and becomes the gold standard of nucleic acid detection. However, the PCR detection technology has the disadvantages of large equipment volume, poor tolerance of a crude sample, long reaction time, difficulty in reaching single copy sensitivity and the like, and is difficult to meet the requirements of modern nucleic acid detection: fast (< 30 min), ultra-sensitivity (1-5 copy/test), direct detection of large-volume crude sample (20-50% reaction volume sample size), field operation, simplified operation, etc. Scientists have attempted to achieve the above objective through isothermal amplification methods in the last 20 years, which include novel isothermal nucleic acid amplification techniques such as RCA (rolling circle amplification), LAMP (loop-mediated isothermal amplification), RPA (restriction enzyme polymerization), NASBA (nucleic acid sequence based amplification), SDA (strand amplification), HDA (helix dependent amplification), TMA (transcription-mediated amplification), and the like.
Among these isothermal amplification techniques, the LAMP method, first reported in 2000, is particularly glaring and interesting, and currently occupies more than 60% of the isothermal detection market. The nucleic acid detection is so dependent on the LAMP technology, and the comprehensive advantages of the LAMP technology cannot be achieved due to the benefit of other constant temperature detection technologies:
1. the reaction speed is extremely high, the optimized primer combination can detect single copy molecules within 15min, and the detection speed is close to or exceeds that of RPA and NASBA;
can reach the hypersensitive detection limit, molecules of 1-5copy can be stably detected, the stability is extremely high, and any other constant temperature technology cannot reach the stable level;
2. the result has diversified interpretation forms, and is suitable for rapid detection of nucleic acid under various environments and conditions. LAMP amplification is used as an end point interpretation mode, and detection results can be observed by naked eyes without any other auxiliary equipment. Results can be observed by naked eyes based on calcein, HNB, red-yellow discoloration and OG orange-green discoloration. LAMP can also be subjected to real-time turbidity analysis by means of a nephelometer. After SYBR Green fluorescent dye is added into a reaction system, a small constant temperature fluorometer can be used for real-time detection. The result interpretation forms can be carried out in non-standard laboratories such as the field, and the required equipment is simple, small and exquisite, low in price and convenient to popularize. Besides, the traditional fluorescent quantitative PCR instrument in a laboratory can also carry out LAMP detection, a SYBR Green, MB probe method or LAMP TaqMan probe method can be adopted, and personnel who often operate PCR detection can be in seamless butt joint without replacing equipment.
3. The omission factor of the RNA virus is low, and because the mutation rate of the RNA virus is high, the influence of individual mutation on PCR amplification in PCR detection is large, and the false negative of the virus omission factor is easily caused. While LAMP recognizes 8 sections of the target gene, individual mutations in these recognition sections have little influence on LAMP amplification and are not easy to generate omission.
4. The reagent is stable and easy to use. Compared with the RPA and NASBA technologies, the LAMP and PCR methods adopt a single enzyme (or double enzyme) system for reaction, the production batch is stable, the reaction enzyme system is high temperature resistant, and the reagent stability is good. The detection reagent only comprises one enzyme mix tube and one primer/probe tube, is convenient to use, and can be conveniently used for high-throughput detection on a fluorescent quantitative PCR machine. And systems such as RPA, NASBA and the like need a plurality of complex enzymes, are strict in proportion, are difficult to stably produce reagents, are harsh in storage and transportation conditions, and are difficult to apply at high flux.
5. The LAMP method has the strongest capability of tolerating impurities. Amplification systems such as PCR, RPA, NASBA and the like all need to be prepared by nucleic acid purification, and in a direct amplification system for crude products, the sample loading amount is small (< 10%), large-volume sample loading cannot be realized, and impurities have great influence on the amplification methods. The LAMP method has tolerance performance of more than 20% on most samples, the content of individual samples can be tolerated to more than 50%, and the LAMP has higher detection rate due to the large sample loading volume.
6. The amplification reaction is synchronous with virus inactivation, and because the LAMP reaction temperature is carried out at the high temperature of 65 ℃, the virus can be inactivated in the reaction process in the direct amplification of virus liquid, thereby reducing the pollution risk of consumables.
7. High specificity, along with continuous improvement of LAMP enzyme, continuous optimization of reaction buffer solution, collocation of probe technology and continuous improvement of primer design concept, the quality of non-specific amplification of LAMP is improved, at present, in an optimized LAMP system, the condition of false positive is fundamentally improved, and the proportion of the false positive is close to or even lower than that of a PCR method.
Disadvantages of LAMP:
(1) The requirements on the primers are particularly high
(2) The amplification product can not be used for clone sequencing, but can only be used for judgment
(3) Because of its strong sensitivity, it is easy to form aerosol, resulting in false positive effect on detection result
(4) And it is difficult to perform various detections
(5) Results based on calcein, HNB, reddish yellow discoloration, OG orange green discoloration can be visually observed. LAMP can likewise be subjected to real-time turbidimetric analysis with the aid of a nephelometer. After SYBR Green fluorescent dye is added into a reaction system, a small constant temperature fluorometer can be used for real-time detection. The result interpretation forms can be carried out in non-standard laboratories such as the field, and the required equipment is simple, small and exquisite, low in price and convenient to popularize. Based on this observation, false positives are prone to occur;
(6) And the detection specificity to mycoplasma is low.
Disclosure of Invention
In view of the defects in the background technology, the invention relates to a mycoplasma LAMP fluorescence detection primer composition and a feline distemper virus LAMP fluorescence detection method thereof, and aims to solve the problems that the primer requirement is high, an amplification product cannot be used for clone sequencing, the sensitivity is low, multiple detections are difficult, the false positive is high, and the specificity to the feline distemper virus is low.
The invention relates to a mycoplasma LAMP fluorescence detection primer composition, which consists of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3 and probes LF, LF1 and LF-Q; the primer sequences are specifically as follows:
F3 5’-CTCAAATGGATGGTGCTATC-3’；
B3 5’-TGGAGCATTATCTCCATCAA-3’；
FIP 5’-CGAGGAACACCAACTTGTCTAGATA-TTAGTTGTTGCTGCAACAGA-3’；
BIP 5’-bio AAGGTGAAGAAGAGATGATTGAACT-TCCGTATTCTGAAAGAAGTGAA-3’；
LF: 5’-AAAGAATGTGTTCACGTGTTTGA-3’；
LF1: 5’-（FAM） GTCAGTGCAGGCTCCCGTAAAGAATGTGTTCACGTGTTTGA（THF）GCATTGGTCCA-3’ （C3-SPACER）；
LF-Q：5’-ACGGGAGCCTGCACTGAC-3’（BHQ1）。
further, the probes LF1 and LF-Q are replaced by LF ', wherein the sequence of LF' is as follows:
LF’ 5’-（BHQ1）ATTGCGGGAGATGAGACCCGCAA（FAM-dT）AAAGAATGTGTTCACGTGTTTGA（THF）GCATTGGTCCA-3’ （C3-SPACER）。
further, the reverse inner primer BIP is provided with a biological lock.
The invention also provides a mycoplasma LAMP fluorescence detection method, which is characterized in that the mycoplasma LAMP fluorescence detection composition is adopted, and the specific detection method is as follows:
12.5ul 2 XBuffer (1.6M betaine, 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 20 mM (NH 4) 2SO4, 4pmol LF, 169mol LF1 and 16pmol LB-Q1ul Bst 2.0 DNA polymerase,8.4 mM MgSO4, 1.2 uM dNTPs, 1ul Endo IV, 2ul template was added and the rest was supplemented to 25ul with water;
placing in a Boy day fluorescent quantitative PCR instrument, reading FAM fluorescence at 40cycles at 60 ℃ for 60s, adding 10ul of product into 190ul of water after reaction, uniformly mixing by oscillation, adding about 60ul of liquid into a nucleic acid test strip by a dropper, and observing for 5 min.
Further, the 169mol LF1 and 16pmol LB-Q were replaced with 16pmol LF'.
The invention has the main beneficial effects that:
1. the specificity of LAMP for detecting the feline calicivirus is greatly improved, and false positive is reduced;
2. multiple LAMP reactions can be carried out;
3. fluorescence and colloidal gold can be detected simultaneously, so that cost and time are saved;
4. based on fluorescence detection, sensitivity is improved.
Drawings
FIG. 1 is a graph showing the amplification curves of the present invention.
Detailed Description
While the embodiments of the present invention will be described and illustrated in detail with reference to the accompanying drawings, it is to be understood that the invention is not limited to the specific embodiments disclosed, but is intended to cover various modifications, equivalents, and alternatives falling within the scope of the invention as defined by the appended claims.
For the convenience of understanding the embodiments of the present invention, the following detailed description will be given by way of example with reference to the accompanying drawings, and the embodiments are not limited to the embodiments of the present invention.
The embodiment 1 of the invention relates to a mycoplasma LAMP fluorescence detection primer composition, which consists of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3 and probes LF, LF1 and LF-Q; the primer sequences are as follows:
F3 5’-CTCAAATGGATGGTGCTATC-3’；
B3 5’-TGGAGCATTATCTCCATCAA-3’；
FIP 5’-CGAGGAACACCAACTTGTCTAGATA-TTAGTTGTTGCTGCAACAGA-3’；
BIP 5’-bio AAGGTGAAGAAGAGATGATTGAACT-TCCGTATTCTGAAAGAAGTGAA-3’；
LF: 5’-AAAGAATGTGTTCACGTGTTTGA-3’；
LF1: 5’-（FAM） GTCAGTGCAGGCTCCCGTAAAGAATGTGTTCACGTGTTTGA（THF）GCATTGGTCCA-3’ （C3-SPACER）；
LF-Q：5’-ACGGGAGCCTGCACTGAC-3’（BHQ1）。
wherein the probes LF1 and LF-Q are replaced by LF ', wherein the sequence of LF' is:
LF’ 5’-（BHQ1）ATTGCGGGAGATGAGACCCGCAA（FAM-dT）AAAGAATGTGTTCACGTGTTTGA（THF）GCATTGGTCCA-3’ （C3-SPACER）。
the reverse inner primer BIP is provided with a biological lock, the biological lock has no influence on fluorescence detection, and meanwhile, a foundation is provided for test strip detection.
The embodiment is provided with a contrast experiment, the probes LF1 and LF-Q are adopted as a reaction system 1, the probe LF' is adopted as a reaction system 2, and the Eva Green fluorescent dye is adopted as a contrast reaction system, and the specific experiment is as follows:
reaction system 1:12.5ul 2 XBuffer (1.6M betaine, 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 20 mM (NH 4) 2SO4, 4pmol LF, 169mol LF1 and 16pmol LB-Q1ul Bst 2.0 DNA polymerase,8.4 mM MgSO4, 1.2 uM dNTPs, 1ul Endo IV, 2ul template was added and the rest was supplemented to 25ul with water;
placing in a Boy day fluorescent quantitative PCR instrument, reading FAM fluorescence at 40cycles at 60 ℃ for 60s, adding 10ul of product into 190ul of water after reaction, uniformly mixing by oscillation, adding about 60ul of liquid into a nucleic acid test strip by a dropper, and observing for 5 min.
Reaction system 2:12.5ul 2 XBuffer (1.6M betaine, 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 20 mM (NH 4) 2SO4, and 0.2% Tween 20), 5pmol F3, 5pmol B3, 40 pmol biotin-FIP primers,40 pmol biotin-BIP,4pmol LF, 169m LF' 1ul Bst 2.0 DNA polymerase,8.4 mM MgSO4, 1.2 uM dNTPs, 1ul Endo IV, 2ul template was added, and the remainder was supplemented with water to 25ul
Placing in a Bori fluorescence quantitative PCR instrument, reading FAM fluorescence at 40cycles at 60 ℃ for 60s, adding 10ul of product into 190ul of water after reaction, uniformly mixing by oscillation, adding about 60ul of liquid into a nucleic acid test strip by a dropper, and observing for 5 min.
Table 1: fluorescent CT value comparison table
Conclusion the CT values in the reaction system 1 and the reaction system 2 both meet the requirement of fluorescence detection, and test paper strip detection can be carried out, so that the cost and the time are saved.
Finally, it should be noted that: the above-mentioned embodiments are only specific embodiments of the present invention, which are used for illustrating the technical solutions of the present invention and not for limiting the same, and the protection scope of the present invention is not limited thereto, although the present invention is described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: those skilled in the art can still make modifications or changes to the embodiments described in the foregoing embodiments, or make equivalent substitutions for some features, within the scope of the disclosure; such modifications, changes or substitutions do not depart from the spirit and scope of the embodiments of the present invention, and they should be construed as being included therein. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (5)
1. A mycoplasma LAMP fluorescence detection primer composition is characterized by comprising a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3 and probes LF, LF1 and LF-Q; the primer sequences are specifically as follows:
F3 5’-CTCAAATGGATGGTGCTATC-3’；
B3 5’-TGGAGCATTATCTCCATCAA-3’；
FIP 5’-CGAGGAACACCAACTTGTCTAGATA-TTAGTTGTTGCTGCAACAGA-3’；
BIP 5’-bio AAGGTGAAGAAGAGATGATTGAACT-TCCGTATTCTGAAAGAAGTGAA-3’；
LF: 5’-AAAGAATGTGTTCACGTGTTTGA-3’；
LF1: 5’-（FAM） GTCAGTGCAGGCTCCCGTAAAGAATGTGTTCACGTGTTTGA（THF）GCATTGGTCCA-3’ （C3-SPACER）；
LF-Q：5’-ACGGGAGCCTGCACTGAC-3’（BHQ1） 。
2. the mycoplasma LAMP fluorescence detection primer composition according to claim 1, wherein the probes LF1 and LF-Q are replaced by LF ', wherein the sequence of LF' is as follows:
LF’ 5’-（BHQ1）ATTGCGGGAGATGAGACCCGCAA（FAM-dT）AAAGAATGTGTTCACGTGTTTGA（THF）GCATTGGTCCA-3’ （C3-SPACER）。
3. the mycoplasma LAMP fluorescence detection primer composition according to claim 1 or 2, wherein the primer composition comprises the following components in percentage by weight: the reverse inner primer BIP is provided with a biological lock.
4. A mycoplasma LAMP fluorescence detection method, which is characterized in that the mycoplasma LAMP fluorescence detection composition of claim 3 is adopted, and the specific detection method is as follows:
12.5ul 2 XBuffer (1.6M betaine, 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 20 mM (NH 4) 2SO4, 4pmol LF, 169mol LF1 and 16pmol LB-Q1ul Bst 2.0 DNA polymerase,8.4 mM MgSO4, 1.2 uM dNTPs, 1ul Endo IV, 2ul template was added and the rest was supplemented to 25ul with water;
placing in a Bori fluorescence quantitative PCR instrument, reading FAM fluorescence at 40cycles at 60 ℃ for 60s, adding 10ul of product into 190ul of water after reaction, uniformly mixing by oscillation, adding about 60ul of liquid into a nucleic acid test strip by a dropper, and observing for 5 min.
5. The Mycoplasma LAMP fluorescence detection method according to claim 4, characterized in that: the 169mol LF1 and 16pmol LB-Q were replaced with 16pmol LF'.
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Application publication date: 20221125 |