Patent Application
20220372464
This research and development result is an automated nucleic acid extraction and qualitative diagnosis. This technology can be used in the field of academic research, clinical pathogen detection, entry-exit inspection operations, and other nucleic acid analysis-related applications. The characteristics of automation and the high-throughput process of this technology are suitable for understaffed units. The simple interpretation method reduces the professional threshold required by operators. In addition, the integrated kit and extraction system realize a single device that can complete nucleic acid extraction and molecular detection at one time.
Since the outbreak of coronavirus disease 2019 (Covid-19), the demand and value of nucleic acid-based pathogen detection technology has increased significantly. Currently, the most common and reliable method to detect the RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is real-time quantitative polymerase chain reaction (qPCR). However, the procedure of qPCR takes a couple of hours and requires well-trained laboratory technicians with expensive equipment. In 2000, Notomi and others further developed the LAMP (loop-mediated isothermal amplification) technology in Japan. Loop-mediated isothermal amplification (LAMP) is an alternate nucleic acid-based detection method that can isothermally produce amplified PCR products resulting in a qualitative diagnosis. This method allows the nucleic acid amplification reaction to be performed at a single temperature (60-65° C.), and the product can yield a thousand times than the traditional PCR. The sensitivity of LAMP can reach a level below 10 copies. In addition, the results of the reaction can be observed through precipitation, fluorescence or color changed, so it is a very convenient and rapid method for nucleic acid testing.
A precise molecular biological testing relies on high-quality and high-efficiency nucleic acid extraction pre-processing. In 2014, the applicant invented the rotating-stirring nucleic acid extraction technology, which automatically extracts the nucleic acid through the magnetic attraction of magnetic bead. The operation time is relatively short and has a low risk of cross-contamination. The extracted nucleic acid can be applied for downstream nucleic acid analysis with real-time PCR (Q-PCR) instrument.
To save the time of extracting nucleic acid, amplifying nucleic acid and analysis of nucleic acid, and further improving the purity of the nucleic acid, combining the extraction and analysis into a one-time automatic process may help. Therefore, a single system and step to achieve automated high-throughput nucleic acid extraction and qualitative method should be needed.
For the purpose of the present disclosure, providing a system for automatic nucleic acid extraction and qualitative analysis, comprising: a magnetic rotary mixer, comprises: a plurality of magnetic rods for generating magnetism, configured to be retractable from the magnetic rotary mixer; a plurality of spin shaft for mounting tips, and the plurality of magnetic rods extend therein; an auto stage, comprises: a plate holder, which allows a plate place thereon; a mixer holder to hold the magnetic rotary mixer over the plate holder; and a heat plate, disposed under the plate holder for heating the plate.
Preferably, the plate holder is horizontally movable.
Preferably, the plate holder is moved by a stepper motor.
Preferably, the mixer holder is vertically movable.
Preferably, the mixer holder is moved by a stepper motor.
Preferably, the magnetic rotary mixer comprises 8 spin shafts.
Preferably, the magnetic rotary mixer further comprises a control panel for controlling a condition of the nucleic acid extraction.
Preferably, the plate has 96 wells.
Preferably, the system further comprises a cover shell.
Preferably, the spin shaft is rotated by a motor.
Preferably, the auto stage comprises a controlled chip with preset programs.
For another purpose of the present disclosure, providing a method for automatic nucleic acid extraction and analysis performed by the above system, comprising: introducing samples, reagents and beads into the plate; conducting a nucleic acid extracting step, the magnetic rotary mixer mixes the samples, the reagents and the beads, and extracts the nucleic acid thereof with the beads; and conducting an analysis step by RT-LAMP, wherein the plate and the magnetic rotary mixer are moved automatically when conducting the nucleic acid extracting step.
Preferably, the plate and the magnetic rotary mixer are moved by the stepper motor.
Preferably, the plate and the magnetic rotary mixer are moved horizontally and vertically respectively.
Preferably, the method further comprises a heating step for controlling the temperature of assay step.
Preferably, the heating step is performed by the heat plate.
Preferably, a reagent of RT-LAMP comprises primer that can combine with the nucleic acid and moderate pH.
Preferably, the reagent of RT-LAMP further comprises pH indicator.
Preferably, the beads are magnetic beads
The automated system disclosed in the present disclosure is designed for mid- to-high throughput nucleic acid extraction application. Specialized spin tips bring in high efficiency in mixing samples, the isolation principle is the collection and transfer of magnetic beads which adsorbs nucleic acid from well to well, and purified DNA and RNA can be obtained after binding, wash, and elution. As such, through using the system for automatic nucleic acid extraction and qualitative analysis disclosed in the present disclosure, user may save more time and labor to obtain a high efficiency nucleic acid extraction and analysis application.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
The present disclosure is depicted by the accompanying drawings, illustrated by embodiments, and described below. The embodiments are implemented in different forms which, however, are not necessarily required to implement or apply the present disclosure. Thus, the different forms of implementation must not be interpreted in a way to limit the embodiments. Features of specific embodiments, steps of a method for constructing and operating specific embodiments, and the sequence of the steps of the method are disclosed hereunder. However, it is also feasible to use any other specific embodiments to achieve identical or equivalent functions and step sequence. Conversely, the embodiments are provided, such that the description hereunder can be thoroughly and completely presented to sufficiently inform persons skilled in the art of the spirit of the present disclosure. Similar reference numerals used in the accompanying drawings denote similar components. Conventional functions or structures are omitted from the description below for the sake of brevity.
Unless otherwise defined, all the technical terms and jargons used hereunder shall have the same meanings as normally understood by persons skilled in the art. If there is any inconsistency between this specification and the comprehension of persons skilled in the art, the definitions-containing specification shall prevail.
Each singular noun used hereunder includes the plural form of the noun without contradicting the context. Each plural noun used hereunder includes the singular form of the noun without contradicting the context. Furthermore, the expression “at least one” and the expression “one or more” used hereunder have the same meaning, and both include one, two, three or more.
The expression “consisting essentially of” used hereunder is for use in defining a composition, method or device, including any materials, steps, features, constituents or components other than what are expressly stipulated. Its restrictive criterion is: the additional materials, steps, features, constituents or components do not significantly affect essential and novel features of an invention claimed. The scope of the expression “consisting essentially of” lies between that of the expression “comprising” and that of the expression “consisting of”.
The relatively broad scope of the present disclosure is defined by numerical ranges and parameters which are approximate for general description. Furthermore, the numerical ranges and parameters inevitably come with standard deviations associated with any examination methods. The aforesaid “about” means that an actual value can be 10%, 5%, 1% or 0.5% greater than or less than a specific value or a limit of a range. Alternatively, the aforesaid “about” means that an actual value falls within an acceptable standard deviation of its mean, depending on the considerations which persons skilled in the art take into account. In addition to the embodiments of the present disclosure, or unless otherwise expressly specified, all the ranges, numbers, numerical values, and percentages (for example, descriptive of the amount of a material in use, a period of time, temperature, operation conditions, numeric proportions, and the like) stated hereunder are each followed by the adverb “about”. Therefore, unless otherwise conversely specified, all the numerical ranges and parameters disclosed hereunder are presented in the form of approximate numerical values and are subject to changes as needed. The numerical values and parameters must be at least interpreted to be applicable to significant figures and general decimal notation. The ranges of the numeral values are each defined with an endpoint and another endpoint or defined as a range between two endpoints. Unless otherwise specified, the ranges of the numeral values disclosed hereunder include their respective endpoints.
There are various nucleic acid extraction methods on the market. However, no matter it is manual or automated extraction methods, additional operating procedures are required if the further nucleic acid analysis is to be performed. At present, the most common way to analyze nucleic acid is the Q-PCR system. The process of Q-PCR includes plate preparation (manual or automatic sampler), sample loading, program setting, analysis progress, and result interpretation. It takes about 2 to 4 hours and the process may increase the possibility of error and contamination. Besides, reagent loading depends on the skill of the operator, the automatic sampler requires additional space, and interpretation of the results requires a trained professional.
To solve the problems mentioned above and achieve the same accuracy (a simpler and rapid nucleic acid analysis), the present disclosure uses an automated nucleic acid extraction instrument developed by the applicant, and a nucleic acid extraction kit containing LAMP reagents to realize one-time automation nucleic acid extraction and analysis technology.
The significant differences between this technology and the existing nucleic acid extraction and analysis methods are:
a. Combine the extraction and analysis into a one-time automatic process.
b. The interpretation is simple and can be observed directly with the naked eye, which does not require extra equipment.
c. The overall operation time is shorter than the real-time PCR system and the sensitivity remains accurate.
Herein after, an automatic nucleic acid extraction and qualitative analysis system 1 of an embodiment of the present disclosure will be describe in detail corresponding with the drawings.
See
In one embodiment of the present disclosure, please refer to the
The plate holder 201 of the auto stage 200 can be movable horizontally. In the automatic extraction and qualitative analysis of the present disclosure, the user only has to prepare the sample from the subjects and the reagents into the corresponding wells of the plate 300, the process will perform automatically. The details of the process will be described later.
To move the plate holder 201 horizontally, the auto stage 200 may comprise a stepper motor. With the stepper motor, the plate holder 201 may move the plate 300 from a well to the next well within a predetermined distance to proceed the nucleic acid extraction process.
On the other hand, for holding the magnetic rotary mixer 100 over the plate holder 201, the auto stage 200 further comprises a mixer holder 203. The mixer holder 203 may hold the magnetic rotary mixer 100 over the plate 300 on the plate holder 201, and move the magnetic rotary mixer 100 vertically with a stepper motor. As such, the magnetic rotary mixer 100 can be moved upward to allow the plate holder 201 move horizontally, and the magnetic rotary mixer 100 can be moved downward to insert the spin tips 103 into the wells.
As shown in
In one embodiment of the present disclosure, as shown in (A) of
As shown in
As shown in
Herein after, the movement of the auto stage 200 will be describe in details.
See
Referring to
The steps mentioned above may be controlled by a controlled chip. User may set desired steps and programs to the control chip, and the auto stage 200 may be operated automatically.
In a preferred embodiment of the present disclosure, the magnetic rotary mixer 100 may have 8 spin shafts 102 and 8 magnetic rods 101, and the plate 300 may have 96 wells with 8 rows and 12 columns. The present disclosure is not limited thereto, the number of spin shaft, magnetic rods and wells of the plate may be predetermined based on needed.
The method of automatic nucleic acid extraction and assay performed by the above mentioned system 1 comprises following steps: introducing samples and reagents into the plate 300; conducting a nucleic acid extracting step, the magnetic rotary mixer 100 mixes the samples and the reagents, and extracts the nucleic acid thereof with beads 104; and conducting an assay step by RT-LAMP, wherein the plate 300 and the magnetic rotary mixer 100 are moved automatically when conducting the nucleic acid extracting step.
In one embodiment of the present disclosure, the plate 300 and the magnetic rotary mixer 100 are moved by the stepper motor to make sure the movement of the plate 300 and the magnetic rotary mixer 100 are in the correct position. In another embodiment of the present disclosure, the method further comprises a heating step for controlling the temperature of assay step performed by the heat plate 202.
In the assay step by RT-LAMP, the reagent may be introduced after the extraction and amplification of the nucleic acid. And the reagent of RT-LAMP may comprise pH indicator, so that user may recognize the result with their naked eyes.
Herein after, the operation of the system 1 and the method will be described with an example.
To test the extraction and detection efficiency of the system in this study, we used synthesized plasmids or commercial pseudovirus (AccuPlex™ SARS-CoV-2 Reference Material, MA, USA) containing the RNA directed against the published CDC and WHO consensus SARS-CoV-2 sequences. The samples were diluted with 1X PBS buffer to proper concentration. Nucleic acids were extracted from 200 μL of samples using a TANBead fully automated magnetic bead operating platform (i.e. the system 1), TANBead Maelstrom™ 8 Autostage (i.e. the auto stage 200) with TANBead Auto Plate (i.e. the plate 300) (Taiwan Advanced Nanotech Inc., Taoyuan City, Taiwan). The auto plate contained 600 μL of lysis buffer, 800 μL of wash buffer, 800 μL of diluted magnetic beads, and 80 μL of elution buffer. The extraction procedure was performed automatically after mounting the tips to magnetic rotary mixer and choosing the program.
For nucleic acid standards used in our assays, present disclosure synthesized two plasmids on pUC57 vector, which containing the open reading frame of the nucleocapsid protein (N) and envelope (E) protein of SARS-CoV2 respectively. The sequences were based on the Genbank accession number NC_045512.2. The region of N gene plasmid included nucleotides 28,273 to 29,533; the region of E gene plasmid included nucleotides 6,245 to 26,472. The plasmids were transformed into E. coli (DH5α) for amplification and isolated by QIAprep Spin Miniprep Kit (MD, USA). The sequences and maps were attached in supplementary materials.
To design RT-LAMP primer sets for detecting of SARS-CoV-2, present disclosure used NEB LAMP Primer Design Tool (https://lamp.neb.com/#!/). Briefly, using the ORF of N or E gene as input sequence and setting normal default parameters. Present disclosure selected three primer sets from predicted results for following assays, the primer sequences were showed in Table. 1. The primers were then synthesized, and the stocks were dissolved in sterilized ddH2O in final 100 μM concentration. Six primers (2 μM F3, 2 μM B3, 16 μM FIP, 16 μM BIP, 4 μ M LF and 4 μM LB) were premixed to generate 10X RT-LAMP primer-mix.
2X colorimetric buffer contained 2.8 mM dNTP, 20 mM (NH4)2SO4, 16 mM MgSO4, 100 mM KCl, 0.2% Tween 20, and 200 μM phenol red, the pH value of reaction buffer was adjusted to 8.1 with 1M KOH. RT-LMAP reactions were prepared in a final 25 μ l volume, each reaction mix contained 12.5 μL of 2X colorimetric buffer, 2.5 μLof 10X RT-LAMP primer-mix, 0.07 μL of Bst 2.0 WarmStart DNA Polymerase (New England Biolabs), 0.5 μL of WarmStart RTx Reverse Transcriptase (New England Biolabs), 2 μL of template, and above components were mixed H2O up to 25 μL. The reactions were incubated at 65° C. for 30 min and observed the color change of the phenol red.
To detect genes of SARS-CoV-2 in this study, quantitative PCR was performed with commercial qPCR mastermix, AllplexTM 2019-nCoV assay (Seegene, Inc., Seoul, South Korea). In short, after thawing all reagents completely, PCR setup was prepared by following reagents: 5 μL of 2019-nCoV MOM, 5 μL of 5X realtime one-step buffer, and 5 μL of real-time one-step enzyme. Mixing PCR setup with inverting and spindown, then 8 μL of nucleic acid sample or positive control was added in pre-mix and ready to perform PCR. The RT-PCR assays were performed under the following protocol: reverse transcription at 50° C. for 20 min and initial denaturation at 95° C. for 15 min, 45 cycles of denaturation at 94° C. for 15 s and annealing at 58° C. for 30s using CFX96∜ Real-Time PCR Detection System (Bio-Rad, USA). The results were considered positive if Ct value is less than 40.
To develop a rapid covid-19 diagnosis system, we designed a workflow including sample collection, automated nucleic acid extraction, and RT-LAMP detection. The nasopharyngeal swab specimen is inserted in a sterile tube contained 2 ml of virus transport medium, for storage or following assay. The auto plate is contains lysis buffer, wash buffer, elution buffer, and RT-LAMP reagents.
In detail, as shown in
In another embodiment, if the user wants to obtain different parts of the nucleic acids for different purposes, for example, one part of nucleic acids for the following RT-LAMP assay and the other parts for other assay, it can be performed by adjusting the times of the elution step. Take two times of the elution step for instance, the fifth row #5 of the wells may be loaded with elution buffer, through controlling the elution time, part of nucleic acids combined with TANBeads 104 may be eluted for other assay, and the rest part of nucleic acids may be bring to the sixth row #6 of the wells for the following RT-LAMP assay as described above.
Viral genomic RNA will be extracted from the swab specimen using a Maelstrom 8 Autostage in 14 min automatically, which is further dissolved in elution buffer and RT-LAMP reagents. After extraction procedure, auto plate will be incubated at 65° C. for 30 min to perform RT-LAMP reaction. A colorimetric result of RT-LAMP can be observed from the bottom of plate. In addition, the sample dissolved in elution buffer can be used for further analysis, such as real-time PCR. In this study, present disclosure aimed to verify the performance of this extraction and detection system.
As the RT-LAMP performed, the extracted RNA may be reverse transcript to DNA and amplified, due to pH indicator inside, turning yellow are considered positive and the wells remaining pink are considered negative, see
To prepare colorimetric RT-LAMP reagent for covid-19 detection, we synthesized three primer sets or nucleocapsid protein (N) gene and envelope (E) gene of SARS-CoV-2 respectively. The target regions were selected according to the genome reference sequence (NC_045512) on NCBI and the primer sets were generated by NEB LAMP Primer Design Tool. The primer sequences used in this study showed in Table. 1. First, we tested the sensitivities of different primer sets by serial dilution of the standard plasmid. A 10-fold dilution was started from 107 to 101 copies per reaction, and the RT-LAMP assays were used 2 μL of the plasmid in a reaction volume of 5 μL. The reactions were assembled on ice and then incubated at 65° C. for 30 min. In N primer set 1 group, 107 to 101 copies turned yellow color, and the non-template control remained pink, as observed before reaction started. In N primer set 2 group, 107 to 102 copies turned to yellow color and 10 copies remained pink, as observed in non-template control. Finally, The N primer set 3 group had a similar result with N primer set 2 (
Next, we tested the detection limit of N/E gene multiplex RT-LAMP by the mix of N and E primer set 1, that based on the results of
The final concentration of two primer set mix is 4.4 μM (same as individual primer set assay), the ratio of N and E primer set is 1 to 1. Similarly, the template used in this assay is N and E plasmid mix with 1 to 1 ratio. Our result showed that 107 to 101 copies turned yellow color, and the non-template control remained pink (see
First, we evaluated the elution efficiency between two elution steps by qPCR assay. It is important to control the two elution steps releases same amount of isolated RNA so the detection results of the two eluents (EB1 and EB2) can be similar. Therefore, the elution time of EB1 and EB2 are different. Similarly, the plasmid controls of E gene from SARS-CoV-2 were ten-fold serially diluted in PBS buffer and extracted by Maelstrom™ 8 Autostage, then eluted into two elution buffers respectively. Next, the E gene plasmid yields in EB1 and EB2 were detected by qPCR to compare two elution steps result respectively.
In A of
As shown in A of
To test the performance of this system, we used various copy number of pseudovirus spike-in nasopharyngeal swab as extraction sample. In addition, we replaced the EB2 with RT-LAMP reagent, allowing to perform the detection of viral RNA after extraction procedure. Moreover, the EB1 were analyzed by qPCR to provide the quantitative Ct value. As shown in
Although automated extraction system brings convenient and fast detection system, the risk of cross contamination during extraction steps between neighboring wells should be considered. In addition, RT-LAMP is highly sensitive thus may be easily contaminated and resulted in false positive. Therefore, we tested whether the cross-contamination to neighbor well exist during TANBead automated extraction and detection. The samples were pseudovirus spike-in nasopharyngeal swab as previous assay, the positive and negative samples were arranged to neighbor well and performed extraction and detection procedure.
As shown in
In conclusion, one embodiment of the present disclosure, we developed an automated extraction and detection system, which contains double elution steps to provide visual RT-LAMP results and ready-to-use samples of RT-qPCR. This system not only reduces hands-on time and time-to-results but also increases the throughput of diagnosis, it may be a useful method during epidemic prevention. However, this system has potential to expand for further applications. Dengue fever, influenza, and Zika virus infection are all important diagnostic targets of infectious diseases. Moreover, based on this system, it is possible to create a high-throughput genotyping system with special designed primers, such as Dengue virus typing, SARS-CoV-2 variants identification, SNP gen-253 otyping, etc.
According to the technical feature described above, the automated system disclosed in the present disclosure is designed for mid-to-high throughput nucleic acid extraction application. Specialized spin tips bring in high efficiency in mixing samples, the isolation principle is the collection and transfer of magnetic beads which adsorbs nucleic acid from well to well, and purified DNA and RNA can be obtained after binding, wash, and elution. As such, through using the system for automatic nucleic acid extraction and qualitative analysis disclosed in the present disclosure, user may save more time and labor to obtain a high efficiency and high accuracy nucleic acid extraction and analysis application.
Although the present invention has been described in terms of specific exemplary embodiments and examples, it can be appreciated by those skilled in the art that changes could be made to the examples described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular examples disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The application incorporates U.S. Provisional Application No. 63/190,393, filed on May 19, 2021, entitled “SINGLE SYSTEM AND STEP TO ACHIEVE AUTOMATED HIGH-THROUGHPUT NUCLEIC ACID EXTRACTION AND QUALITATIVE METHOD” as reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63190393 | May 2021 | US |
