Method for blocking LAMP (loop-mediated isothermal amplification) non-specific amplification by using nano material

Abstract

The invention relates to the field of biological detection, in particular to a method for blocking LAMP (loop-mediated isothermal amplification) nonspecific amplification by using a nano material and application of the method to transgenic corn detection. A high-accuracy real-time fluorescent quantitative detection method is constructed by using a loop-mediated isothermal amplification technology and Au (at) Pt NPs or AuNPs; a transformant specific primer group is designed according to a connecting region sequence of a 5'end of a transgenic corn DBN 9936 genome and an exogenous gene insertion segment, before an amplification system is prepared, nano material-SNA (NPs-SNA) is thermally dried by using Au (at) Pt or AuNPs and the primer group, nucleic acid amplification and real-time fluorescence monitoring are carried out, non-specific amplification of LAMP can be blocked on the basis, and the detection result is accurate. The high-accuracy detection of the specific sequence of the transgenic corn DBN 9936 transformant is realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of loop-mediated isothermal amplification for detection of transgenic corn, and particularly relates to detection of transgenic corn DBN9936 by NPs-SNA based on loop-mediated isothermal amplification. BACKGROUND

[0002] Transgenic component determination is divided into three methods based on DNA, foreign protein, and biological determination. Among them, the DNA determination method mainly uses specific primers (probes) for DNA amplification, and determines the amplification product by end-point PCR or real-time fluorescent PCR. End-point PCR is performed according to the standard PCR mode, and the result is determined by electrophoresis band or fluorescence signal after amplification. It is commonly used for qualitative detection. Real-time fluorescent quantitative PCR is divided into qualitative and quantitative two types. Quantitative detection is based on the fluorescence signal labeled on the primer or probe, and the number of DNA molecules amplified in each cycle is determined by observing the activation of the fluorescence signal of the DNA amplification product in real time. Qualitative detection can be further divided into two types: (1) comparing the number of cycles required for the fluorescence signal to reach the defined threshold, if it is higher than the defined threshold before the given PCR cycle number, the determination result is positive; (2) dye-embedding method identifies the amplification product by checking the melting temperature (Tm value) or by other suitable ways. PCR cycle includes three key stages: denaturation of target DNA, annealing and extension, and a typical PCR detection needs 30-40 cycles, which takes several minutes to several hours. The shortcomings of complex operation process, dependence on bulky equipment such as thermal cycler, long time consumption, and the need for professional operation limit its application in point of care testing (POC).

[0003] The development of loop-mediated isothermal amplification (LAMP) nucleic acid detection eliminates thermal cycling, simplifies the detection process, reduces energy consumption, and improves detection sensitivity, greatly promoting the development of on-site detection. However, the problem of false positives is a common problem in LAMP-based nucleic acid detection. In the process of on-site identification of the purity and authenticity of transgenic corn seeds, the test results based on LAMP will directly affect the researchers' judgment of the purity and authenticity of the batch of seed samples, thereby leading to wrong decisions. Therefore, it is particularly important to carry out research on blocking false positives in LAMP amplification detection to reduce the false positive rate of large-scale transgenic corn seed detection and improve the scientificity and accuracy of decision-making. SUMMARY

[0004] The present application aims to provide a method for blocking non-specific amplification to overcome the shortcomings of the prior art.

[0005] The first aspect of the present application provides a method for LAMP non-specific amplification blocking using NPs-SNA, characterized in that the method comprises: (1) sample pretreatment: DNA extraction is performed on the biological sample to obtain a DNA template; (2) dilute all the required reagents to the appropriate concentration; (3) construct NPs-SNA; (4) add the required reagents for LAMP reaction, then perform amplification reaction, and monitor the fluorescence curve in real time, and identify the results according to whether there is an amplification curve within the optimized reaction time.

[0006] Further, the construction method of NPs-SNA in step (3) is as follows: heat the nanomaterial aqueous solution and the FIP, BIP, F3, and B3 primer solutions required for the reaction in a dry metal bath thermostat at 85-95°C until the water is completely evaporated, then add water to dissolve, and the amount of water added is the sum of the amount of dd H2O, the total amount of primers, and the amount of nanomaterial aqueous solution.

[0007] Further, the nanomaterial is gold nanoparticles AuNPs or gold core platinum shell nanoparticles Au@Pt NPs; Further, the nanomaterial is Au@Pt NPs, the particle size of the Au@Pt NPs is selected to be 5-15 nm, and the addition amount of the Au@Pt NPs is 20 ng-500 ng, preferably, the final concentration of the addition is 0.8-20 ng / µL.

[0008] Further, the nanomaterial is AuNPs, the particle size of the AuNPs is 10-15 nm, and the addition amount of the AuNPs is 3.16-6.32 nM.

[0009] The second aspect of the present application provides the use of the method of the first aspect in the detection of transgenic plants.

[0010] Further, the transgenic plant is transgenic corn; preferably, the transgenic corn is transgenic corn DBN 9936.

[0011] Further, the primers used in the LAMP are selected from one group of the following A-E groups: The primer group A is Primer 1-F3, Primer 1-B3, Primer 1-FIP, and Primer 1-BIP, respectively as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4; The primer group B is Primer 2-F3, Primer 2-B3, Primer 2-FIP and Primer 2-BIP, respectively as shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; The primer group C is Primer 3-F3, Primer 3-B3, Primer 3-FIP and Primer 3-BIP, respectively as shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12; The primer group D is Primer 4-F3, Primer 4-B3, Primer 4-FIP and Primer 4-BIP, respectively as shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; The primer group E is Primer 5-F3, Primer 5-B3, Primer 5-FIP and Primer 5-BIP, respectively as shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20; Preferably, the LAMP primer group is selected from the primer group A.

[0012] Further, the LAMP detection system comprises long primers FIP and BIP, short primers F3 and B3, a deoxynucleotide solution mixture, magnesium sulfate, 10x isothermal amplification buffer solution, Bst2.0 WarmStart DNA Polymerase and SYBR Green I. Further, the NPs-SNA is prepared by using a nano material and long primers and short primers. Further, the LAMP reaction condition is that the reaction is carried out at 65 ℃ for 90 min in a buffer system with a total volume of 25 μL.

[0013] The third aspect of the present application is to provide a LAMP detection composition of the method of the first aspect, characterized in that the composition comprises NPs-SNA, which is prepared from the nano material and the LAMP primer group.

[0014] Further, the composition further comprises other reagents required for LAMP detection.

[0015] The fourth aspect of the present application provides the composition in the application of the second aspect, characterized in that the composition comprises NPs-SNA, and the NPs-SNA is prepared by the nanomaterial and the LAMP primer set.

[0016] Further, the composition further comprises other reagents required for LAMP detection.

[0017] Compared with the prior art, the present application has the following advantages and beneficial effects: 1. The present application realizes the blocking of LAMP non-specific amplification by introducing NPs-SNA into the LAMP system. 2. The detection limit of the method is 0.01 ng / µL. 3. The method realizes high-accuracy detection of transgenic corn DBN 9936 based on LAMP. BRIEF DESCRIPTION OF DRAWINGS

[0018] The accompanying drawings, which are included to provide a further understanding of the embodiments of the present application and constitute a part of the application, illustrate embodiments of the present application and do not limit the present application. In the drawings: Figure 1 False positive analysis in transgenic corn DBN 9936 LAMP primer screening and mass amplification (A. Comparison of fluorescence quantitative LAMP amplification efficiency of five groups of primers; B. Mass amplification results of Primer 1 in 8 groups of negative samples); Figure 2 Au@Pt NPs (A. TEM of Au@Pt; XPS of Au@Pt); Figure 3 Au@Pt-SNA LAMP performance evaluation and optimization (A. Real-time fluorescence quantitative result chart of Au@Pt-SNA LAMP; B. Comparison of amplification efficiency of traditional LAMP, Au@Pt LAMP and Au@Pt-SNA LAMP for positive samples; C. Optimization of Au@Pt-SNA LAMP system); Figure 4 Au@Pt-SNA LAMP detection performance evaluation (A. Sensitivity; B. Specificity); Figure 5For Au@Pt-SNA LAMP universality evaluation (A. Transgenic corn 12-5, Bt11 x GA21 traditional LAMP fluorescence quantitative results; a. Transgenic corn 12-5 traditional LAMP fluorescence quantitative results figure; b. Transgenic corn Bt11 x GA21 traditional LAMP fluorescence quantitative results figure; B. Transgenic corn 12-5, Bt11 x GA21 Au@Pt-SNA LAMP fluorescence quantitative results; a. Transgenic corn 12-5 Au@Pt-SNA LAMP fluorescence quantitative results figure; b. Transgenic corn Bt11 x GA21 Au@Pt-SNA LAMP fluorescence quantitative results figure; C. False positive blocking effect of Au@Pt-SNA LAMP in the specific detection of transgenic corn DBN 9936 transformation event (primer: Primer 2, 3, 4); Figure 6 For NPs-SNA LAMP material universality (A. False positive blocking effect of AuNPs-SNA LAMP in the specific detection of transgenic corn DBN 9936 transformation event; a. AuNPs UV-Vis spectrum; b. Influence of AuNPs-SNA LAMP on the amplification efficiency of transgenic corn DBN 9936 genomic DNA, wherein the abscissa represents the concentration of AuNPs, not the concentration of AuNPs added to the LAMP system; c. False positive blocking effect of AuNPs-SNA LAMP; B. Performance evaluation of 5 nm, 15 nm Au@Pt-SNA LAMP in the specific detection of transgenic corn DBN 9936 transformation event; a. Influence of 5 nm, 8 nm Au@Pt constructed Au@Pt-SNA LAMP on the amplification efficiency of transgenic corn DBN 9936 genomic DNA; b. False positive blocking effect of 5 nm, 8 nm Au@Pt constructed Au@Pt-SNA LAMP. DETAILED DESCRIPTION

[0019] The embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings. The embodiments described below by referring to the drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application. Modifications or replacements of the method, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application.

[0020] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those which would be understood by one of ordinary skill in the art to which the application pertains. All patents and publications referred to in this application are incorporated by reference in their entirety.

[0022] Principle design and verification of the method of blocking LAMP non-specific amplification by nanomaterials (1) Principle design and verification The present application combines NPs-SNA with LAMP and uses it to detect transgenic corn DBN 9936. The principle is that, (1) during the heat drying process, the nucleic acid secondary structure anneals, and then NPs are constructed into NPs-SNA by binding with part of the inner primer, which reduces the concentration of free primers in the system, which is conducive to reducing the formation of primer dimers and other unfavorable structures; (2) NPs-SNA has a local high ion concentration effect, which is easy to aggregate cations in the system, and interacts with Bst 2.0 WarmStart DNA Polymerase, thereby affecting the "ion microenvironment (Mg 2+ ) " to realize dynamic regulation of the enzymatic reaction rate, inhibiting the activity of Bst 2.0 WarmStart DNA Polymerase at room temperature, and then blocking the occurrence of non-specific amplification during the system configuration process, and improving the fidelity of the enzyme.

[0023] The present application relates to the construction of NPs-SNA: Heat the nanomaterial aqueous solution together with all the primers required for the reaction in a 90℃ dry metal bath thermostat until the water is completely evaporated (12-15min), then add water to dissolve, and the amount of water added is the sum of the amount of dd H2O in the original system, the total amount of primers and the amount of nanomaterial aqueous solution.

[0024] The present application involves real-time fluorescence quantification in the verification process, and the specific steps are: Add other reagents required for LAMP reaction to the prepared NPs-SNA solution. The LAMP amplification reaction is carried out in a real-time fluorescence quantitative PCR instrument, the reaction temperature is 65℃, the reaction time is 120 min, and the fluorescence value is recorded once per minute.

[0025] The present application relates to the synthesis of Au@Pt, and the specific steps are: In a 50 ml three-necked flask, add HAuCl4 aqueous solution (10 mg / ml), H2PtCl6 aqueous solution (10 mg / ml) and ultrapure water (45 ml), heat the heating jacket to boiling, keep for 10 min, then add 200 μl of trisodium citrate aqueous solution (114.1 mg / ml), react for 30 min, after the reaction, at room temperature, place on a magnetic stirrer, cool to room temperature. In order to quantify the Au@Pt solution, the cooled Au@Pt solution is placed at -80°C overnight, then placed in a freeze dryer for vacuum freeze drying, the freeze-dried Au@Pt powder is weighed, and an appropriate amount of water is added to prepare an Au@Pt aqueous solution. The characterization of 8 nm Au@Pt NPs is shown in Figure 2 During the preparation of Au@Pt, the addition amount of HAuCl4 aqueous solution (10 mg / ml) and H2PtCl6 aqueous solution (10 mg / ml) is shown in Table 2.

[0026] The present application relates to the synthesis of AuNPs, and the specific steps are as follows: All glassware required during the experiment is soaked in aqua regia overnight, then washed several times with ultrapure water until clean. Add 100 mL of 1 mM chloroauric acid (HAuCl4) solution to a round-bottom flask, keep stirring slowly and heat to boiling, then immediately add 10 mL of 38.8 mM trisodium citrate solution which has been filtered with a 0.22 μM pore size membrane in advance, keep heating for 15-20 min, the final solution turns into stable wine red, stop heating, keep stirring slowly until the temperature decreases to room temperature, store the colloidal gold solution in the dark at 4°C for later use (2) Primer selection Based on the real-time fluorescent quantitative loop-mediated isothermal amplification (Real-time LAMP) technology, the DNA genome (10 ng / μL) of transgenic maize DBN 9936 was used as a template, and the reaction was carried out according to the traditional LAMP system described in claim 9. Five groups of transgenic event-specific LAMP primers designed in Table 1 were screened, and Figure 1 is the Real-time fluorescent quantitative LAMP result graph of the five primers. By comparing and contrasting the amplification kinetics parameters (Cq value) and end-point fluorescence intensity, it was found that the amplification efficiency of Primer 1 was significantly better than that of other primers, indicating that it could efficiently recognize and amplify the specific sequence of the transgenic maize DBN 9936 transformation event. Moreover, Primer 1-5 did not appear false positive results caused by non-specific amplification in the experimental range, therefore, Primer 1 was selected as the primer for subsequent experiments.

[0027] However, as Figure 1The false positive rate of Primer 1 in a large number of negative sample verifications is 37.5%, which is caused by non-specific amplification in the system. This contradictory phenomenon shows that although the transformation event-specific LAMP primer can accurately target the target sequence, it still has the risk of non-specific amplification. In high-throughput applications, measures need to be taken to block non-specific amplification to ensure the reliability of transgenic component detection. Since the non-specific amplification phenomenon in LAMP reaction is affected by random factors such as random formation of primer dimers and transient binding of non-target regions of the template, in the follow-up experiment, 8 groups of parallel repeats were set for the negative control group and 3 groups of parallel repeats were set for the positive control group to effectively reduce false positive interference and ensure detection reliability through statistical significance analysis.

[0028] Performance of the detection method in Example 2 (1) Specificity Under the optimal reaction conditions, the specificity of Au@Pt-SNA LAMP for transgenic corn DBN 9936 was tested. The genomes of transgenic corn 12-5, 2A-7, and Bt11xGA21 were extracted, and then the genome concentration was diluted to 10 ng / µL. Primer-1 was used as the Au@Pt-SNA LAMP primer group, and 20 ng / µL 8 nm Au@Pt was used to construct Au@Pt-SNA. Then, real-time fluorescence quantitative monitoring was performed on 10 ng / µL of transgenic corn 12-5, 2A-7, and Bt11xGA21 genomes, respectively. The results are shown in Figure 4 As shown in -B, Au@Pt-SNA LAMP has good specificity, and only when the transgenic corn DBN 9936 genome is present in the system, there is a real-time fluorescence quantitative curve.

[0029] (2) Sensitivity In order to obtain the qualitative detection limit of Au@Pt-SNA LAMP for detecting transgenic corn DBN 9936, the extracted transgenic corn DBN 9936 genome was diluted to 10, 1, 0.1, and 0.01, respectively, and then used for Au@Pt-SNA LAMP detection. The results are shown in Figure 4 As shown in -A, in the experiment, the minimum Cq value of the target without adding is 122, indicating that when the reaction time exceeds 120 min, false positive results are likely to occur, so it is recommended that the detection time does not exceed 120 min. Under this condition, the qualitative detection limit of Au@Pt-SNA LAMP for detecting transgenic corn DBN 9936 is 0.01 ng / µL.

[0030] Evaluation of the detection universality in Example 3 (1) Evaluation of the universality of non-specific amplification blocking for different targets To explore the blocking effect of Au@Pt-SNA LAMP method on non-specific amplification when using different primer groups for LAMP detection, the universality of the detection target and the universality of different primer groups for the same target were explored, and the experimental results are shown in Figures 2-6 , where 5-A is the real-time fluorescence quantitative result diagram of traditional LAMP for transgenic corn 12-5, Bt11xGA21, both of which have non-specific amplification, 5-B is the real-time fluorescence quantitative result diagram of Au@Pt-SNA LAMP for transgenic corn 12-5, Bt11xGA21, and the results show that within the experimental range, the Au@Pt-SNA LAMP group does not appear non-specific amplification curve.

[0031] (2) Evaluation of non-specific blocking universality of different primer groups for the same target Figure 5 -C is the performance evaluation of Au@Pt-SNA LAMP in the process of detecting transgenic corn DBN 9936 using Primer 2, Primer 3, and Primer 4 primer groups. The results show that, as shown in Figure 5 -C-b, Au@Pt-SNA LAMP can block non-specific amplification in the process of detecting DBN 9936 using Primer 2, Primer 3, and Primer 4, and as shown in Figure 5 -C-a, the efficiency of specific amplification is not significantly inhibited, on the contrary, in the process of using Primer 4 for transgenic corn DBN 9936 transformant event specific diagnosis, the specific amplification efficiency of Au@Pt-SNA LAMP is significantly improved compared with traditional LAMP.

[0032] (3) Exploring the universality of nano materials in NPs-SNA LAMP system for non-specific amplification blocking Subsequently, the universality of nano materials in NPs-SNA LAMP was explored, including material type universality and material particle size universality, and the experimental nano materials included: 13 nm AuNPs, 5 nm Au@Pt, and 15 nm Au@Pt. As shown in Figure 6 , first, the prepared AuNPs were characterized by UV-Vis spectroscopy, and the results are shown in Figure 6-A-a shows that the prepared AuNPs have a maximum absorption peak near 520 nm, and the corresponding absorbance is 0.427. According to the Lambert-Beer law, the concentration of the concentrated AuNPs is 15.8 nM. Subsequently, AuNPs-SNA LAMP was constructed using different concentrations of AuNPs, and the blocking effect of non-specific amplification was evaluated, as shown in Figure 6 -A-b shows that the constructed AuNPs-SNA LAMP has no significant effect on the amplification efficiency of the transgenic maize DBN 9936 transformant event-specific detection based on Primer 1, and, as shown in Figure 6 -A-c shows that in the range of 3.16-6.32 nM, the non-specific amplification inhibition effect gradually increases with the increase of AuNPs, and 6.32 nM AuNPs is sufficient to completely block non-specific amplification. However, non-specific amplification occurs at 12.64 nM AuNPs, which may be due to the high concentration of AuNPs, the low surface nucleic acid density of the constructed AuNPs-SNA after heat drying, and the poor stability.

[0033] Subsequently, the non-specific amplification blocking effect of 5 nm and 15 nm Au@Pt constructed Au@Pt-SNA LAMP was explored, and the results are shown in Figure 6 -B shows that, Figure 6 -B explores the effect of 5 nm and 15 nm Au@Pt addition amount on the amplification efficiency in the specific detection of transgenic maize DBN 9936 transformant event, and the results show that 15 nm Au@Pt has a certain inhibitory effect on the amplification efficiency when added at 20 ng / µL. Figure 6 -B evaluates the non-specific amplification blocking effect of 5 nm and 15 nm Au@Pt at different addition amounts, and the results show that 5 nm Au@Pt added at 0.8 ng / µL is sufficient to completely block non-specific amplification, which may be due to the smaller particle size, the larger specific surface area at the same addition amount, and the stronger restriction on FIP and BIP. However, 15 nm Au@Pt cannot completely block false positives in the experimental concentration range.

[0034] Table 1 LAMP primer set sequence

[0035] Table 2 Preparation of Au@Pt with different particle sizes

[0036] Table 3 Traditional LAMP reaction system

[0037] Table 4 Au@Pt-SNA LAMP reaction system

Claims

1. A method for LAMP non-specific amplification blocking using nanomaterials, characterized in that, The method comprises: (1) sample pretreatment: extracting DNA from the biological sample to obtain a DNA template; (2) diluting all required reagents to appropriate concentrations; (3) constructing NPs-SNA; (4) adding LAMP reaction required reagents to the system and then performing amplification reaction, real-time monitoring fluorescence curve, and identifying results according to whether there is an amplification curve within the optimized reaction time; The construction method of the NPs-SNA in step (3) is as follows: heating a nanomaterial aqueous solution and LAMP reaction required FIP, BIP, F3 and B3 primer solutions in a dry metal bath constant temperature device at 85-95°C until water is completely evaporated, and then adding water to redissolve, wherein the amount of added water is the sum of the amount of original dd H2O, the total amount of primers and the amount of added nanomaterial aqueous solution.

2. The method of claim 1, wherein, The nanomaterial is Au@Pt NPs, the particle size of the Au@Pt NPs is selected to be 5-15 nm, and the final concentration of the added Au@Pt NPs is 0.8-20 ng / µL.

3. The method of claim 1, wherein, The nanomaterial is AuNPs, the particle size of the AuNPs is 10-15 nm, and the amount of added AuNPs is 3.16-6.32 nM.

4. Use of the method of any one of claims 1-3 in detection of transgenic plants.

5. Use according to claim 4, characterized in that, The transgenic plant is transgenic corn, and the transgenic corn is transgenic corn DBN 9936.

6. Use according to claim 5, characterized in that, The primers used in the LAMP are selected from one group of A-E groups: Primer group A is Primer 1-F3, Primer 1-B3, Primer 1-FIP and Primer 1-BIP, which are respectively shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4; Primer group B is Primer 2-F3, Primer 2-B3, Primer 2-FIP and Primer 2-BIP, which are respectively shown as SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; Primer group C is Primer 3-F3, Primer 3-B3, Primer 3-FIP and Primer 3-BIP, which are respectively shown as SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12; Primer group D is Primer 4-F3, Primer 4-B3, Primer 4-FIP and Primer 4-BIP, which are respectively shown as SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16; Primer set E is Primer 5-F3, Primer 5-B3, Primer 5-FIP, Primer 5-BIP, respectively as shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:

20.

7. Use according to claim 6, characterized in that, The detection system of the LAMP includes: long primers FIP and BIP; short primers F3 and B3; a deoxynucleotide solution mixture; magnesium sulfate; 10x isothermal amplification buffer solution; Bst2.0 WarmStart DNA Polymerase; and SYBR Green I.

8. The LAMP detection composition for the method according to any one of claims 1 to 3, characterized by, The composition includes NPs-SNA, which is prepared from the nanomaterial and the LAMP primer set.

9. The LAMP assay composition for use according to any one of claims 4 to 7, characterized in that, The composition includes NPs-SNA, which is prepared from the nanomaterial and the LAMP primer set.

10. The composition of claim 8 or 9, characterized in that, The composition further includes other reagents required for LAMP detection.

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