Colorimetric constant-temperature circular nucleic acid amplification system
By developing a colorimetric constant temperature ring nucleic acid amplification (LAMP) system, including colorimetric LAMP reaction mixture and extraction-free lysis buffer, problems such as long turnover time and complex equipment in existing nucleic acid detection technologies have been solved, and rapid, sensitive and dedicated detection of viral gene substances have been achieved.
Patent Information
- Application Number
- CN202410193008.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-12-29
- Filing Date
- 2024-02-21
- Publication Date
- 2025-07-01
AI Technical Summary
The existing nucleic acid detection technology has problems such as long turnover time, large equipment size, high cost, high complexity and high sample purity requirements, which limits its application in home use and in use by untrained personnel.
A colorimetric constant temperature ring nucleic acid amplification (LAMP) system, including a colorimetric LAMP reaction mixture and an extraction-free lysis buffer, was developed, allowing the entire mixture to be freeze-dried for temperature stability, and nucleic acid amplification and detection through an extraction-free direct lysis buffer.
Fast, sensitive and dedicated detection of viral gene substances is achieved, shortening diagnosis time, reducing equipment costs and complexity, and allowing application in home use and untrained personnel.
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Figure CN120230830A_ABST
Abstract
Description
Technical Field
[0001] This application relates to a constant temperature loop-mediated isothermal amplification (LAMP) system, especially a colorimetric LAMP system.
Background Art
[0002] Disease diagnosis generally can be divided into disease antigen detection, disease nucleic acid detection or even antibody detection. Taking COVID-19 as an example, currently two main types of tests are used: molecular tests (also known as nucleic acid tests (NAT)) and antigen rapid tests (ART). Nucleic acid tests using qPCR or qRT-PCR technology are adopted as the gold standard in some countries because they are more accurate than antigen rapid tests. However, a trade-off is that most traditional nucleic acid test results take about 24 hours to obtain, and some even take longer because they need to be sent to an external laboratory, while antigen rapid tests can give results in just 15 minutes, and the cost of producing antigen rapid test kits is also much lower. The problem is that antigen rapid tests are not as sensitive as nucleic acid tests (about 1000 times less sensitive), so they are more likely to give false negative and false positive results. Therefore, compared with nucleic acid tests, antigen rapid tests have lower specificity, sensitivity and reliability.
[0003] Many types of nucleic acid tests have been developed, but there are still some drawbacks. For example, the turnaround time of traditional nucleic acid tests is relatively long, thus reducing the effectiveness of preventing pathogen transmission through early diagnosis and protective measures. In addition, traditional nucleic acid test devices are large in size, costly, and complex, making them not conducive to use by untrained personnel and for home settings. Another type of nucleic acid test with a short turnaround time and lower capital investment was developed in 2000, called loop-mediated isothermal amplification (LAMP). The design of LAMP allows users to detect the amplification status through a turbidimeter or a fluorescence detector, both of which require a large amount of capital investment, thus increasing the cost as a home device. Another type of nucleic acid test uses colorimetric detection of nucleic acid amplification, in which the reaction mixture includes an enzyme and a halochromic agent, allowing users to distinguish the sample status by observing color changes with the naked eye. However, since the reagent formulation contains high concentrations of cryoprotective components, such as betaine and glycerol, etc., to prevent the reagent from being freeze-dried, it means that the reagent must be transported by cold chain to prevent enzyme degradation, so the opportunity as a home device is also reduced due to considerations of reagent stability. Although freeze-dried compositions have been developed, the enzyme and primer parts in the PCR components are freeze-dried separately, which also increases the preparation procedure and lengthens the preparation time.
[0004] In addition to the need for freeze-drying to achieve reagent stability for home settings, another problem with nucleic acid tests is sample purity. The main drawback of nucleic acid tests is that the sample input for testing needs to have high purity, which involves complete nucleic acid extraction and purification. Although various methods for high-quality nucleic acid extraction have been developed, there may be a risk of nicking the nucleic acid during the extraction process, and the chance of affecting the sensitivity of LAMP is increased due to the longer amplicon size. In addition, users need to spend time purchasing the correct extraction kit before using the test, which also prolongs the diagnostic time and limits the diagnosis to be carried out only in laboratories equipped with complex institutional settings.
[0005] Therefore, it is necessary to provide a nucleic acid test system to address the challenges encountered in the prior art.
Summary of the Invention
[0006] One objective of this application is to provide a colorimetric loop-mediated isothermal amplification (LAMP) system that can quickly, sensitively, and specifically diagnose viral genomic material with the naked eye.
[0007] Another object of the present application is to provide a colorimetric LAMP reaction mixture that allows the user to lyophilize the entire mixture to achieve temperature stability.
[0008] Another object of the present application is to provide an extraction-free lysis buffer compatible with the colorimetric LAMP reaction mixture.
[0009] To achieve the above object, the present application provides a colorimetric LAMP system, including a colorimetric LAMP reaction mixture and an extraction-free lysis buffer. The colorimetric LAMP reaction mixture can be lyophilized in its entirety and includes: a primer set for amplifying a target sequence, a strand displacement polymerase, and deoxynucleoside triphosphates; a pH indicator dye with a concentration range of 0.08 to 0.3 mM; and a cryoprotective sugar with a concentration range of 1 to 10% (w / v), where the cryoprotective sugar is selected from the group consisting of trehalose, raffinose, dextran, mannitol, and mixtures thereof. The extraction-free lysis buffer includes: potassium chloride with a concentration range of 10 to 50 mM; ammonium sulfate with a concentration range of 10 to 50 mM; and a detergent with a concentration range of 0.5 to 6% (w / v), where the detergent is selected from the group consisting of 2-ethylhexan-1-ol; 2-methyloxirane; ethylene oxide (CAS No. 64366-70-7), secondary alcohol ethoxylate (CAS No. 84133-50-6), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (CAS No. 9002-93-1), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethan-1-ol (CAS No. 9002-93-1), ethyl {2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}dodecanoate (CAS No. 9005-64-5), and mixtures thereof. After the lyophilized colorimetric LAMP reaction mixture is rehydrated with the extraction-free lysis buffer, nucleic acid amplification and detection can be performed.
[0010] In one embodiment, the colorimetric LAMP reaction mixture further includes reverse transcription for converting viral genomic RNA into complementary DNA.
[0011] In one embodiment, the pH indicator dye is phenol red or neutral red.
[0012] In one embodiment, the volume of the colorimetric LAMP reaction mixture is less than 10 μl.
[0013] In one embodiment, the colorimetric LAMP reaction mixture includes 2 to 7.5% (w / v) of trehalose and 2 to 7.5% (w / v) of raffinose.
[0014] In one embodiment, the colorimetric LAMP reaction mixture comprises 2 to 7.5% (w / v) trehalose, 2 to 7.5% (w / v) raffinose, 1 to 2.5% (w / v) glucan, and 1 to 5% (w / v) mannitol.
[0015] In one embodiment, the colorimetric LAMP reaction mixture further comprises a magnesium salt in a concentration range of 4 to 12 mM.
[0016] In one embodiment, the detergent in the extraction-free lysis buffer is 0.5 to 6% (w / v) 2-ethylhexan-1-ol; 2-methyloxirane; oxirane (CAS No. 64366-70-7).
[0017] In one embodiment, the detergent in the extraction-free lysis buffer is 0.5 to 6% (w / v) secondary alcohol ethoxylate (CAS No. 84133-50-6).
[0018] In one embodiment, the detergent in the extraction-free lysis buffer is 0.5 to 6% (w / v) 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (CAS No. 9002-93-1).
[0019] In one embodiment, the detergent in the extraction-free lysis buffer is 0.5 to 6% (w / v) 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethan-1-ol (CAS No. 9002-93-1).
[0020] In one embodiment, the detergent in the extraction-free lysis buffer is 0.5 to 6% (w / v) ethyl {2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}dodecanoate (CAS No. 9005-64-5). [Description of the Drawings]
[0021] Figure 1A Showing the color change performance of colorimetric LAMP using phenol red as a pH indicator dye. Figure 1B Showing the color change performance of colorimetric LAMP using neutral red as a pH indicator dye. Figure 2A Showing the lyophilized beads in a microcentrifuge tube. Figure 2B Showing the lyophilized cakes in an 8-well strip tube. Figure 3 Showing the color change after rehydration and amplification of the lyophilized beads. Figure 4 Showing the performance comparison between a fresh colorimetric LAMP reaction mixture and a lyophilized bead colorimetric LAMP reaction mixture. Figure 5 Show the performance comparison between commercially available RT-LAMP and freeze-dried cake colorimetric LAMP. Figure 6 Show the performance comparison between freeze-dried beads and freeze-dried cakes using the same set of samples. Figure 7A Show the real-time stability test of the colorimetric LAMP reaction mixture at room temperature, Figure 7B Show the endpoint RFU (relative fluorescence unit), Figure 7C Show the Cq value of the stability test. Figures 8A to 8E Show the performance of various detergents in lysing the live virus HCoV-OC43. Figures 9A to 9C Show the performance of the direct lysis buffer against different viruses. Figure 10A And Figure 10B Show the real-time stability and performance of the direct lysis buffer against extracted RNA and live virus, respectively.
Detailed Description of the Invention
[0022] Some embodiments demonstrating the features and advantages of the present application will be described in detail in the following description. It should be understood that the present application can have various variations in different aspects, all of which do not depart from the scope of the present application, and the descriptions and drawings therein are for illustrative purposes in essence and not for limiting the present application.
[0023] The present application uses a modified isothermal loop-mediated isothermal amplification (LAMP) that uses a pH indicator to develop color, also known as colorimetric LAMP, for rapid, sensitive, and specific diagnosis of viral genomic materials by the naked eye. The present application provides a detection kit and method for detecting viral genomic materials using LAMP. This method includes a reverse transcription step and an isothermal amplification step, where reverse transcription converts viral genomic RNA into complementary DNA (cDNA), and isothermal amplification uses a primer set to amplify the cDNA.
[0024] Specifically, the present application provides a colorimetric LAMP system. The colorimetric LAMP system includes a colorimetric LAMP reaction mixture, and an extraction-free lysis buffer, also known as a direct lysis buffer. The colorimetric LAMP reaction mixture allows the user to lyophilize the entire mixture to achieve temperature stability, and the extraction-free lysis buffer is compatible with the colorimetric LAMP reaction mixture. The present application relates to a formulation for producing a lyophilized colorimetric LAMP reaction mixture in bead form or cake form, and a protocol for direct nucleic acid extraction without purification. The formulations of the colorimetric LAMP reaction mixture and the direct lysis buffer are described in detail below.
[0025] The colorimetric LAMP reaction mixture includes enzymes and materials for nucleic acid amplification, a pH indicating dye, and a sugar. The enzyme for nucleic acid amplification is a strand-displacing polymerase, and the materials for nucleic acid amplification include a primer set and deoxyribonucleoside triphosphates (dNTPs) for amplifying a target sequence. The pH indicating dye provides a color change under visible light, thereby allowing the user to distinguish the amplification results. The sugar is a lyoprotectant sugar at a specific concentration, which can protect the colorimetric LAMP reaction mixture (especially the enzyme) from degradation during lyophilization. Through the presence of a suitable lyoprotectant sugar, the colorimetric LAMP reaction mixture of the present application can be lyophilized all-in-one into a lyophilized ready-to-use reagent after the lyophilization process.
[0026] The direct lysis buffer includes specially selected chaotropic salts and detergents for ideal nucleic acid extraction. The direct lysis buffer can lyse viruses and directly extract nucleic acids, such as respiratory viruses including influenza virus A (FluA), influenza virus B (FluB), respiratory syncytial virus (RSV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and human coronavirus OC43 (HCoV-OC43), but not limited thereto. The lyophilized colorimetric LAMP reaction mixture can be rehydrated with the direct lysis buffer and prepared for nucleic acid amplification and detection..
[0027] In one embodiment, the colorimetric LAMP reaction mixture includes a primer set for amplifying a target sequence, a strand displacement polymerase and deoxynucleoside triphosphates (dNTPs), a pH indicator dye in a concentration range of 0.08 to 0.3 mM, and a lyoprotectant sugar in a concentration range of 1 to 10% (w / v). The lyoprotectant sugar is selected from the group consisting of trehalose, raffinose, dextran, mannitol, and mixtures thereof.
[0028] In one embodiment, the colorimetric LAMP reaction mixture further includes a reverse transcriptase for converting viral genomic RNA into complementary DNA.
[0029] In one embodiment, the pH indicator dye is phenol red or neutral red, but not limited thereto.
[0030] In one embodiment, the volume of the colorimetric LAMP reaction mixture can range from 8 to 20 μl. In particular, the volume of the colorimetric LAMP reaction mixture can be reduced to less than 10 μl, such as 8 to 10 μl, which is beneficial for reducing the volume of the lyophilized beads or cakes compared to the standard of 20 μl.
[0031] In one embodiment, the colorimetric LAMP reaction mixture includes 2 to 7.5% (w / v) of trehalose and raffinose as lyoprotectants, and the volume of the colorimetric LAMP reaction mixture is 9.5 μl.
[0032] In one embodiment, 60% (w / v) of D-(+)-trehalose dihydrate and 30% (w / v) of D-(+)-raffinose pentahydrate are prepared and dissolved in molecular grade water, and the final concentration of the cryoprotectant in the colorimetric LAMP reaction mixture is 2 to 7.5% (w / v) each.
[0033] In one embodiment, the colorimetric LAMP reaction mixture includes 2 to 7.5% (w / v) of trehalose, 2 to 7.5% (w / v) of raffinose, 1 to 2.5% (w / v) of dextran, and 1 to 5% (w / v) of mannitol as cryoprotectants, and the volume of the colorimetric LAMP reaction mixture is 9.5 μl.
[0034] In one embodiment, the colorimetric LAMP reaction mixture further includes a magnesium salt in the concentration range of 4 to 12 mM, such as magnesium sulfate (MgSO4). The magnesium salt helps to target the primers and tightly bind to the target sequence.
[0035] In one embodiment, the colorimetric LAMP reaction mixture further includes an intercalating fluorescent dye, such as dye, for quality control or detection using fluorescent LAMP.
[0036] An exemplary formulation of the colorimetric LAMP reaction mixture for SARS-CoV-2 detection is shown in Table 1, and distilled water is further added to a total volume of 9.5 μl. Table 1
[0037] In addition to the development of the colorimetric LAMP assay, this application also includes the development of a colorimetric LAMP-friendly direct lysis buffer. The direct lysis buffer enables direct amplification after virus lysis and release of genomic material into the buffer without further purification, thus reducing the total detection time.
[0038] This application provides a formulation of a direct lysis buffer for releasing viral genomic material, and the direct lysis buffer with genomic material can be directly added to the above colorimetric LAMP reaction mixture without further purification.
[0039] The direct lysis buffer of this application uses an optimized buffer composition including detergents and LAMP-tolerant chemicals to achieve virus lysis and amplification.
[0040] In one embodiment, the direct lysis buffer of the present application comprises potassium chloride (KCl) in a concentration range of 10 to 50 mM, ammonium sulfate [(NH4)2SO4] in a concentration range of 10 to 50 mM, and a detergent in a concentration range of 0.5 to 6% (w / v). The detergent is selected from the group consisting of 2-ethylhexan-1-ol; 2-methyloxirane; oxirane (trade name Ecosurf TM EH-9, CAS number 64366-70-7), secondary alcohol ethoxylate (trade name Tergitol TM Type 15S-7 or Type 15S-9, CAS number 84133-50-6), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, trade name X-100, CAS number 9002-93-1), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethan-1-ol (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethan-1-ol, trade name CAS number 9002-93-1), {2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate ({2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate, trade name 20, CAS number 9005-64-5) and mixtures thereof. This composition constitutes a direct lysis buffer that is friendly to the colorimetric LAMP reaction.
[0041] An exemplary formulation of the direct lysis buffer for use with the above freeze-dried colorimetric LAMP reaction mixture is shown in Table 2, and the pH value of the direct lysis buffer is 8.0 to 9.0, which can be adjusted with potassium hydroxide (KOH) and hydrochloric acid (HCl). Table 2
[0042] In one embodiment, the direct lysis buffer comprises 10 to 50 mM of potassium chloride, 10 to 50 mM of ammonium sulfate, and 0.5 to 6% (w / v) of EcosurfTM EH-9. This composition constitutes a direct lysis buffer friendly to the colorimetric LAMP reaction and can release the genomic material of SARS-CoV-2 or HCoV-OC43.
[0043] In one embodiment, a premix of 100 to 500 mM potassium chloride and ammonium sulfate is prepared and diluted with molecular water. The pH of the premix can be adjusted to pH 8.1 to 8.8 with 10N KOH and 1N HCl ("N" represents equivalent concentration or normality). Then, the premix with pH 8.5 is used to prepare a direct lysis buffer with 10% (w / v) Ecosurf TM EH-9 to form a direct lysis buffer comprising 10 to 50 mM potassium chloride, 10 to 50 mM ammonium sulfate, and 0.5 to 6% (w / v) Ecosurf TM EH-9.
[0044] In one embodiment, the direct lysis buffer comprises 10 to 50 mM potassium chloride, 10 to 50 mM ammonium sulfate, and 0.5 to 6% (w / v) Tergitol TM Type 15S-7.
[0045] In one embodiment, the direct lysis buffer comprises 10 to 50 mM potassium chloride, 10 to 50 mM ammonium sulfate, and 0.5 to 6% (w / v) Tergitol TM Type 15S-9.
[0046] In one embodiment, the direct lysis buffer comprises 10 to 50 mM potassium chloride, 10 to 50 mM ammonium sulfate, and 0.5 to 6% (w / v) of X-100.
[0047] In one embodiment, the direct lysis buffer comprises 10 to 50 mM potassium chloride, 10 to 50 mM ammonium sulfate, and 0.5 to 6% (w / v) of
[0048] In one embodiment, the direct lysis buffer comprises 10 to 50 mM potassium chloride, 10 to 50 mM ammonium sulfate, and 0.5 to 6% (w / v) of 20.
[0049] The applications of the colorimetric LAMP reaction mixture and the direct lysis buffer of the present application will be illustrated by examples below.
[0050] Example 1 demonstrates the comparison of fluorescence LAMP and colorimetric LAMP using SARS-CoV-2 reference RNA. The colorimetric LAMP assay uses the above-described colorimetric LAMP reaction mixture and pH indicator dyes such as phenol red and neutral red, and an intercalating fluorescent dye is added to the formulation for the fluorescence LAMP assay. Figure 1A shows the color change efficacy of colorimetric LAMP using phenol red as the pH indicator dye, while Figure 1B shows the color change efficacy of colorimetric LAMP using neutral red as the pH indicator dye. Figure 1A and Figure 1B the upper part of shows the amplification curve of the fluorescence LAMP assay, while Figure 1A and Figure 1B the lower part of shows the visual results of the colorimetric LAMP assay. For phenol red, the color of the reaction mixture of positive samples turns orange / yellow after amplification, while the color of the reaction mixture of negative samples remains pink. For neutral red, the color of the reaction mixture of positive samples turns red after amplification, while the color of the reaction mixture of negative samples turns yellow. As shown in Figure 1A and Figure 1B , the color change of colorimetric LAMP is consistent with the amplification results of fluorescence LAMP. The colorimetric LAMP reaction time is 20 minutes, corresponding to 40 Cq in the amplification cycle.
[0051] The colorimetric LAMP reaction mixture of the present application uses an optimized buffer composition, so a rapid diagnostic turnaround time of 20 minutes can be achieved, and this is all achieved without affecting the detection sensitivity of the SARS-CoV-2 kit.
[0052] Furthermore, the colorimetric LAMP reaction mixture of the present application is a freeze-dried ready-to-use formulation, which allows the colorimetric LAMP reaction mixture to be freeze-dried and remain stable at room temperature. This formulation can be freeze-dried in a strip tube to form a lyo-cake, or a lyo-bead can be formed using a bead dispenser, without changing the composition.
[0053] Example 2 demonstrates the freeze-dried colorimetric LAMP reaction mixture of the present application. Figure 2A shows the lyo-beads in a microcentrifuge tube, Figure 2B shows the lyo-cakes in an 8-well strip tube. The lyo-beads and lyo-cakes use the same freeze-drying formulation with the cryoprotectant concentration in the above examples.
[0054] Example 3 demonstrates the rehydration of the freeze-dried colorimetric LAMP reaction mixture. As described above, the freeze-dried colorimetric LAMP reaction mixture can be easily rehydrated through direct lysis buffer. In this example, 9.5 μl of the colorimetric LAMP reaction mixture is freeze-dried into lyo-beads, and phenol red is used as the pH indicator dye.Figure 3 Show the color change of the rehydrated and amplified lyophilized beads. First, rehydrate the lyophilized beads with the direct lysis buffer, and its color shows a return to the original pink, and the color of the positive samples turns yellow or orange after amplification. In addition, using the fluorescence signal as a performance indicator, it can also be seen that the lyophilized colorimetric LAMP reaction mixture (lyophilized beads or lyophilized cakes) shows similar performance after rehydration with the direct lysis buffer.
[0055] Example 4 demonstrates the performance comparison between the fresh and lyophilized bead colorimetric LAMP reaction mixtures using SARS-CoV-2 reference RNA, and an intercalating fluorescent dye is added to the colorimetric LAMP reaction mixture to use the fluorescence signal as a performance indicator. In this example, the performance of the lyophilized bead colorimetric LAMP reaction mixture is compared with that of the freshly prepared liquid colorimetric LAMP reaction mixture, and phenol red is used as the pH indicator dye. The tested virus concentrations are 10,000 cp / ml (10 times the limit of detection (10x LoD)), 3,000 cp / ml (3x LoD), and 0 cp / ml. Figure 4 Show the performance comparison between the fresh colorimetric LAMP reaction mixture and the lyophilized bead colorimetric LAMP reaction mixture. The results show that only the colors of the positive samples with Cq values of 13.99, 13.47, 18.21, and 15.92 turn yellow, while the other samples remain pink. Therefore, comparable sensitivity can be observed between the fresh reagent and the lyophilized beads. In addition, compared with the fresh reagent, the fluorescent Cq value of the lyophilized beads is 2 Cq earlier.
[0056] Therefore, compared with the freshly prepared colorimetric LAMP reaction mixture, the formulation of the colorimetric LAMP reaction mixture of the present application can lyophilize the colorimetric LAMP reaction mixture without loss of the performance of the colorimetric LAMP detection after rehydration.
[0057] Example 5 demonstrates the performance comparison between the lyophilized colorimetric LAMP reaction mixture of the present application and a commercially available RT-LAMP reagent using SARS-CoV-2 reference RNA, and an intercalating fluorescent dye is added to the colorimetric LAMP reaction mixture to use the fluorescence signal as a performance indicator. In this example, the colorimetric LAMP reaction mixture includes 5% (w / v) trehalose and 5% (w / v) raffinose as cryoprotectants, and the lyophilized cake colorimetric LAMP reaction mixture is rehydrated with the direct lysis buffer. Figure 5Show the performance comparison between commercially available RT-LAMP and freeze-dried cake colorimetric LAMP. The results show that the freeze-dried cake colorimetric LAMP reaction mixture used with direct lysis buffer exhibits performance comparable to that of commercially available RT-LAMP reagents and sensitivity comparable to that of RT-LAMP reagents, where the detection rates for the reference RNA at 37.5 copies / reaction are all 100%.
[0058] Example 6 demonstrates the performance comparison between freeze-dried beads and freeze-dried cake colorimetric LAMP reaction mixtures, and an intercalating fluorescent dye is added to the colorimetric LAMP reaction mixture to use the fluorescence signal as a performance indicator. In this example, the freeze-dried beads and freeze-dried cakes use the same lyophilization formulation, which adopts the cryoprotectant concentration of Example 5. The tested virus concentrations are 10000 cp / ml (10x LoD) and 3000 cp / ml (3x LoD). Figure 6 Show the performance comparison between freeze-dried beads and freeze-dried cakes using the same set of samples. The results show that the freeze-dried bead and freeze-dried cake colorimetric LAMP reaction mixtures exhibit comparable performance.
[0059] Example 7 demonstrates the stability of the freeze-dried colorimetric LAMP reaction mixture of the present application, and an intercalating fluorescent dye is added to the colorimetric LAMP reaction mixture to use the fluorescence signal as a performance indicator. In this example, the formulation of the present application is also used for SARS-CoV-2 detection, and both low-concentration samples (0.0011 TCID50) and high-concentration samples (0.018 TCID50) are tested. Figure 7A Show the real-time stability test of the colorimetric LAMP reaction mixture at room temperature, Figure 7B Show the endpoint RFU (relative fluorescence unit), Figure 7C Show the Cq value of the stability test. The results show that the performance of the freeze-dried beads for low-concentration and high-concentration samples remains stable until the 13th week, and the endpoint RFU also shows that a stable signal is obtained until the 13th week. Therefore, the freeze-dried colorimetric LAMP reaction mixture of the present application can be stored at room temperature until at least the 13th week without loss of its performance.
[0060] Example 8 demonstrates the performance of direct lysis buffers prepared using different detergents and used with the freeze-dried colorimetric LAMP reaction mixture, and an intercalating fluorescent dye is added to the colorimetric LAMP reaction mixture to use the fluorescence signal as a performance indicator. In this example, the direct lysis buffer includes a detergent, which are respectively X-100, Ecosurf TM EH-9, Tergitol TM Type 15S-7, Tergitol TM Type 15S-9, or 20, and the preferred concentration range of each detergent is 0.5 to 6% (w / v). In this example, a direct lysis buffer was used to lyse the live virus HCoV-OC43, which can be representative of SARS-CoV-2 as both are human coronaviruses. Figures 8A to 8E Shows the performance of various detergents in lysing the live virus HCoV-OC43. It can be clearly seen from the figure that each of the selected detergents is capable of releasing viral genomic material and facilitating the LAMP reaction. Therefore, compared to using water (dH2O) as the lysis buffer, the direct lysis buffer of the present application can indeed release genomic material from the virus.
[0061] Example 9 demonstrates the performance of the direct lysis buffer used with a freeze-dried colorimetric LAMP reaction mixture on various live virus strains, and an intercalating fluorescent dye was added to the colorimetric LAMP reaction mixture to use the fluorescence signal as a performance indicator. In this example, the live viruses HCoV-OC43, FluA H3N2, and FluB were respectively incorporated into the direct lysis buffer, then mixed with the colorimetric LAMP reaction mixture and amplified. Figures 9A to 9C Shows the performance of the direct lysis buffer on different viruses. As shown in the figure, compared to lysing the virus with water, lysing the virus with the direct lysis buffer can detect an earlier Cq, indicating that the direct lysis buffer can lyse live viruses and release more genomic material for amplification, thereby improving the sensitivity of the colorimetric LAMP detection. In addition, in the HCoV-OC43 samples lysed with the direct lysis buffer, the lowest input concentration could be detected 100%, while in the HCoV-OC43 samples lysed with water, only a 50% detection rate was observed.
[0062] Example 10 demonstrates the stability and lysis efficiency of the direct lysis buffer used with a freeze-dried colorimetric LAMP reaction mixture on RNA or live viruses, and an intercalating fluorescent dye was added to the colorimetric LAMP reaction mixture to use the fluorescence signal as a performance indicator. In this example, to determine the stability and performance of the direct lysis buffer, the live virus FluA H3N2 and RNA extracted from the virus FluA H3N2 were both used for testing. The RNA sample concentrations used to test the performance of the direct lysis buffer were 32xLoD (7.5 TCID50 / reaction) and 4x LoD (0.9375 TCID50 / reaction), while the live virus sample concentrations were 8xLoD (60 pfu) and 4x LoD (30 pfu). Figure 10A and Figure 10BThe real-time stability and performance of the direct lysis buffer for extracting RNA and live virus were shown separately. As shown in the figure, the performance at week 32 was comparable to that at week 6, where the ΔCq of the extracted RNA was less than 2 Cq, and the ΔCq of the live virus was less than 3 Cq. Therefore, the performance of the direct lysis buffer remained comparable to that of freshly prepared buffer until week 32 of preparation. The stability study continued until week 60, and the results also showed that the direct lysis buffer could be stored at room temperature until week 60 without loss of its performance.
[0063] In summary, the present application provides a colorimetric LAMP system for detecting viral genomic materials with high sensitivity, high specificity, and short reaction time. Specifically, the present application provides a formulation of a colorimetric LAMP reaction mixture, which can achieve rapid and efficient LAMP amplification, detect through color change, and retain the functionality of the main components, especially enzymes, in the colorimetric LAMP reaction mixture during lyophilization. A direct lysis buffer system compatible with the colorimetric LAMP reaction mixture is also provided. Therefore, the colorimetric LAMP system of the present application has the following advantages.
[0064] The direct lysis buffer can effectively lyse and release the nucleic acids of the virus in nasal samples. At the same time, the buffer can maintain the integrity of the nucleic acids for downstream nucleic acid amplification. Usually, the time required for commercially available column extraction or magnetic bead extraction methods may be about 30 minutes or longer. In contrast, the direct lysis buffer of the present application requires less than 2 minutes, preferably less than 1 minute, and has a comparable lysis efficiency for the target virus. In addition, the direct lysis buffer of the present application can effectively remove inhibitors in the samples without the purification steps commonly used in other nucleic acid separation procedures, helping to form a buffer system with better sensitivity.
[0065] Furthermore, the components with preferred concentrations in the direct lysis buffer are not only compatible with the colorimetric LAMP reaction but also can promote the colorimetric LAMP reaction. In addition, the direct lysis buffer is adjusted to an appropriate starting pH value and uses an appropriate buffer system to maintain the initial sample pH value, while ensuring that the buffer is pH-sensitive at the start of the LAMP reaction to obtain the pH signal transition of positive samples. Therefore, the direct lysis buffer is a pH-sensitive and stable buffer system, which is used for signal discrimination.
[0066] In particular, the colorimetric LAMP reaction mixture can be freeze-dried in its entirety into freeze-dried beads or cakes. The formulation of the colorimetric LAMP reaction mixture is specially prepared to incorporate both enzymes and primers in a single freeze-dried bead / cake, thereby avoiding the different freeze-drying processes required by the prior art. The selected lyoprotectant sugar allows the reagent to be freeze-dried in a smaller volume (less than 10 μl), resulting in smaller-sized freeze-dried beads / cakes with a morphologically stable spherical shape and performance comparable to that of liquid reagents. In addition, the freeze-dried reagent can be rehydrated within seconds and does not generate bubbles after rehydration, avoiding any interference during the optical signal detection process. The freeze-dried reagent has excellent room temperature stability and can be stored, transported, and used at room temperature after rehydration with a direct lysis buffer.
[0067] Moreover, the colorimetric LAMP system has a short turnaround time. The colorimetric LAMP reaction can be completed within 20 minutes, which is faster than the PCR detection system that takes about 1 hour for RNA detection. The colorimetric LAMP system has distinguishable positive amplification color changes, and its signals can be detected by the naked eye or any other optical detection system.
[0068] Although the present invention has been described in detail by the above embodiments and can be variously modified by those skilled in the art, all such modifications do not depart from the scope of protection as defined in the appended claims.
Claims
1. A colorimetric constant temperature circular nucleic acid amplification (LAMP) system, comprising: A colorimetric LAMP reaction mixture can be lyophilized in its entirety and comprises: A primer set, a strand displacement polymerase, and deoxynucleoside triphosphates for amplifying a target sequence; a pH indicator dye in a concentration range of 0.08 to 0.3 mM; and A lyophilized protective sugar in a concentration range of 1 to 10% (w / v), wherein the lyophilized protective sugar is selected from the group consisting of trehalose, raffinose, dextran, mannitol and a mixture thereof; and An extraction-free lysis buffer, including: Potassium chloride in concentrations ranging from 10 to 50 mM; ammonium sulfate in a concentration range of 10 to 50 mM; and A cleaning agent in a concentration range of 0.5 to 6% (w / v), wherein the cleaning agent is selected from the group consisting of 2-ethylhexan-1-ol; 2-methylethylene oxide; ethylene oxide (CAS No. 64366-70-7), secondary alcohol ethoxylate (CAS No. 84133-50-6), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (CAS No. 9002-93-1), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethan-1-ol (CAS No. 9002-93-1), {2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}dodecanoic acid ethyl ester (CAS No. 9005-64-5) and mixtures thereof; The freeze-dried colorimetric LAMP reaction mixture can be rehydrated with the extraction-free lysis buffer to perform nucleic acid amplification and detection.
2. The colorimetric LAMP system as claimed in claim 1, wherein the colorimetric LAMP reaction mixture further comprises a reverse transcription for converting a viral genomic RNA into a complementary DNA.
3. The colorimetric LAMP system of claim 1, wherein the pH indicator dye is phenol red or neutral red. The colorimetric LAMP system of claim 1 , wherein the volume of the colorimetric LAMP reaction mixture is less than 10 μl. 5 . The colorimetric LAMP system of claim 1 , wherein the colorimetric LAMP reaction mixture comprises 2 to 7.5% (w / v) trehalose and 2 to 7.5% (w / v) raffinose.
6. The colorimetric LAMP system of claim 1, wherein the colorimetric LAMP reaction mixture comprises 2 to 7.5% (w / v) trehalose, 2 to 7.5% (w / v) raffinose, 1 to 2.5% (w / v) dextran, and 1 to 5% (w / v) mannitol.
7. The colorimetric LAMP system of claim 1, wherein the colorimetric LAMP reaction mixture further comprises a magnesium salt in a concentration range of 4 to 12 mM.
8. The colorimetric LAMP system of claim 1, wherein the detergent in the extraction-free lysis buffer is 0.5 to 6% (w / v) of 2-ethylhexan-1-ol; 2-methyloxirane; ethylene oxide (CAS No. 64366-70-7).
9. The colorimetric LAMP system of claim 1, wherein the detergent in the extraction-free lysis buffer is 0.5 to 6% (w / v) of secondary alcohol ethoxylate (CAS No. 84133-50-6).
10. The colorimetric LAMP system of claim 1, wherein the detergent in the extraction-free lysis buffer is 0.5 to 6% (w / v) 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (CAS No. 9002-93-1).
11. The colorimetric LAMP system of claim 1, wherein the detergent in the extraction-free lysis buffer is 0.5 to 6% (w / v) of 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethan-1-ol (CAS No. 9002-93-1).
12. The colorimetric LAMP system of claim 1, wherein the detergent in the extraction-free lysis buffer is 0.5 to 6% (w / v) of ethyl {2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}dodecanoate (CAS No. 9005-64-5).