WO2022201181A1

WO2022201181A1 - RT-LAMP ASSAY FOR DETECTION OF HUMAN β-ACTIN HOUSEKEEPING GENE

Info

Publication number: WO2022201181A1
Application number: PCT/IN2022/050183
Authority: WO
Inventors: Shyam Sundar NANDI; Upendra P. LAMBE; Sonali Ankush Sawant; Trupti GOHIL; Jagadish Mohan DESHPANDE
Original assignee: Indian Council of Medical Research
Current assignee: Indian Council of Medical Research
Priority date: 2021-03-24
Filing date: 2022-03-03
Publication date: 2022-09-29
Anticipated expiration: 2023-09-24
Also published as: BR112023018559A2

Abstract

The present invention relates to primers and a method for detecting internal control (IC) gene (Beta actin) by using a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. This IC can be used in combination with various other RT-LAMP based diagnostic assays. The IC helps as an indicator for quality check of sample collection, nucleic acid extraction and quality of reaction. The present invention also provides kit for internal control detection method established by the invention.

Description

RT-LAMP ASSAY FOR DETECTION OF HUMAN β-ACTIN HOUSEKEEPING GENE TECHNICAL FIELD: The present disclosure relates generally to the field of molecular biology. Particularly, the present disclosure provides novel primers and rapid loop mediated isothermal amplification method for detection of internal control (IC) gene which can be employed along with other RT-LAMP assays for the detection of pathogens. BACKGROUND ART: Point-of-care diagnostic devices permit physicians to obtain rapid, inexpensive information crucial to providing effective patient care. For diagnosis of an infectious disease, gene amplification devices theoretically can provide rapid and sensitive identification while eliminating the need for pathogen cultures and/or large biological sample size. A rapid, specific genetic amplification device also permits the detection of specific alleles or other genetic risk factors that facilitate individualized tailoring of therapeutic regimens. Methods for gene amplification include polymerase chain reaction (PCR), strand displacement amplification (SDA), ligase chain reaction (LCR), and transcription mediated amplification (TCA). However, these technologies are limited by the number of multiple reagents with varying stability for such amplification as well as a reliance on expensive equipment. Currently, the rRT-PCR (Real-time PCR) is being used for the confirmatory diagnosis of many pathogens. However, the rRT-PCR based assays still face issues such as long detection period, complex operation, and sophisticated instrument requirement. These problems are overcome by the primers and method provided in the present invention. Loop-mediated isothermal amplification technology(LAMP) is a strand displacement kind of nucleic acids amplification technique. Reverse transcription loop-mediated isothermal amplification RT-LAMP is based on LAMP augmentation detection of RNA, with addition of a specialized reverse transcriptase. It carries out simultaneously reverse transcription and nucleic acid amplification under constant temperature (65 ℃) condition. SUMMARY: The present invention relates to primers and a method for detecting internal control (IC) gene (Beta actin) by using a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. This IC can be used in combination with various other RT- LAMP based diagnostic assays. The IC helps as an indicator for quality check of sample collection, nucleic acid extraction and quality of reaction. The present invention also provides kit for internal control detection method established by the invention. BRIEF DESCRIPTION OF THE DRAWINGS: These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings wherein: Fig.1 shows Interpretation of the RT-LAMP assay for housekeeping gene-beta actin. Fig. 2 shows Parallel testing results of 34 clinical samples performed using RT-LAMP assay and ICMR-NIV 2019nCoV Assay Kit V 3.1. DETAILED DESCRIPTION: For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the figures and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting. The terminology and structure employed herein is for describing, teaching and illuminating some embodiments and their specific features and elements and does not limit, restrict or reduce the spirit and scope of the invention. Definitions For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person skilled in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below. The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”. Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference. The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purposes of exemplification only. Functionally- equivalent products and methods are clearly within the scope of the disclosure, as described herein. Aspects and embodiments of the present invention will be described below in detail with reference to the accompanying drawings. The present invention relates to primers and a method for detecting internal control (IC) gene (Beta actin) by using a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. This IC can be used in combination with various other RT- LAMP based diagnostic assays. The IC helps as an indicator for quality check of sample collection, nucleic acid extraction and quality of reaction. RT-LAMP technology is adopted, and multiple sets of primers were designed for the human β-actin gene. The internal control detection by RT-LAMP can be employed for quality check of samples while doing field testing of various pathogens using RT-LAMP. In the present invention, the RT-LAMP primers were designed for β-Actin housekeeping gene. The internal control gene is included to check the quality of the collected swab samples in the assay. This housekeeping gene may be included in any RT-LAMP assay. In an embodiment, the present invention provides nucleotide sequences which target internal control (IC) gene (Beta actin), wherein said nucleotide sequences have SEQ ID NOs.1, 2, 3, 4 and 5. In another embodiment, said SEQ ID No. 1 and SEQ ID No. 2 are outer primer pairs; SEQ ID No.1 is forward outer primer β-Actin F3 and SEQ ID No.2 is backward outer primer β-Actin B3. In yet another embodiment, said SEQ ID No.3 and SEQ ID No.4 are inner primer pairs; SEQ ID No.3 is forward inner primer β-Actin FIP and SEQ ID No.4 is backward inner primer β-Actin BIP. In one embodiment, said SEQ ID No.5 is forward loop primer β-Actin LF. In another embodiment, the present invention provides a primer mixture which targets internal control (IC) gene (Beta actin) based on loop mediated isothermal amplification (LAMP) technique comprising a sum total of 5 primers: (i) outer primer pairs comprising forward outer primer β-Actin F3 represented as SEQ ID NO: 1 and backward outer primer β-Actin B3 represented as SEQ ID NO: 2; (ii) inner primer pairs comprising forward inner primers β-Actin FIP represented as SEQ ID NO: 3 and backward inner primer β-Actin BIP represented as SEQ ID NO: 4; (iii) loop forward primer β-Actin LF represented as SEQ ID NO: 5; wherein the primers having GC content ranging from 40-65%, free energy up to –4 kcal/ mol or less at 3’ and 5’ ends of the primers and the primer melting temperature (T_m) ranging from 59 - 61°C for forward and backward primers and 64 - 66°C for loop primers. In another embodiment, three pairs of primers (F3 and B3): (FIP and BIP): (LF/ LB) are at a molar ratio of 0.2:1.6:0.4 respectively. In one more embodiment, the present invention provides a kit for detecting the internal control gene, wherein said kit comprising the primers mixture as explained above. In yet another embodiment, the present provides a rapid loop mediated isothermal amplification method for detection of internal control (IC) gene in a sample employed along with other RT-LAMP assays comprising the steps of: i. extracting RNA from a sample; ii. converting the RNA sample in complimentary DNA by using RT- LAMP technology and amplifying said DNA through LAMP amplification method involving the primer system comprising: iii. outer primer pairs: forward outer primers β-Actin F3 represented as SEQ ID NO: 1 and backward outer primer β-Actin B3: SEQ ID NO: 2; iv. inner primer pairs: forward inner primers β-Actin FIP represented as SEQ ID NO: 3 and backward inner primer β-Actin BIP represented as SEQ ID NO: 4 and loop forward primer β-Actin LF represented as SEQ ID NO: 5; v. detecting the amplified DNA by change of colour which is considered positive reaction when change of color is from pink to yellow. In yet another embodiment, said method is conducted at 65˚C for 30 minutes. The test assay of the present application find application at check points such as at airports, ports and immigration points. Advantages of the innovation: ● The assay can be performed using heating block (65 ± 1° C). ● The time required for the assay is only 40 minutes. ● The results can be interpreted visually; no other sophisticated instruments are required for interpretation of results. ● Highly skilled or technical person is not required to perform this assay. ● It is cost effective and less time-consuming technology. ● This assay could be used in point-of-care RT-LAMP assays outside of the diagnostic laboratory. EXAMPLES: The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art. Example 1: For the detection of Beta actin gene, the RT-LAMP assay reaction is set up. The reaction tubes are incubated at 65˚C for 30 minutes. A color change from pink to yellow is recorded as positive and no color change is recorded as negative. RT-LAMP: RT-LAMP (Reverse Transcription-Loop-mediated Isothermal Amplification) method can synthesize cDNA from template RNA and apply LAMP technology to amplify and detect them. As the template is RNA sample, in addition to the reagents of DNA amplification (primers, DNA polymerase with strand displacement activity, substrates, etc.), reverse transcriptase is added to the reaction mixture. After mixing and incubating at a constant temperature between 60-65°C, amplification and detection can be carried out in a single step. Primers: RT-LAMP uses multiple forward and reverse (backward) primers and one or two loop primers. LAMP uses the RTx reverse transcriptase that gets activated above 45^oC and modified Bst 2.0 DNA polymerase having strand displacement activity. RT-LAMP reactions can therefore be carried out at a single incubation temperature (isothermal). Primers B2 and B3 initiate cDNA synthesis. FIP and BIP are specialized primers consisting of two parts, F2/F1c and B2/B1c, respectively. F2 and B2 bind with the template strand to initiate amplification process while F1c and B1c sequences serve as overhangs which help loop formation as RT-LAMP reaction continues. The short distance between the F2 and F1c (and B2/ B1c) helps formation of a loop structure within the amplicon. The loop primers increase the number of initiation points for DNA synthesis by binding complementarily to the single stranded loops and increase the pace of amplification. For this innovation, novel sets of primers are designed for β actin housekeeping genes. The online software Primer Explorer V5 was used for the designing purpose. Characteristic properties of primers: The four key factors in the LAMP primer design are the melting temperature (T_m), stability at the 3’ and 5’ end of each primer (Delta G), GC content and ability to form secondary structures. T_m: T_m is calculated by using the Nearest-Neighbor method. This method is presently considered to be the method that predicts the Tm value closest to the actual value. The Tm for each region is designed to be about 65°C (64 - 66°C) for F1c and B1c, about 60°C (59 - 61°C) for F2, B2, F3, and B3, and about 65°C (64 - 66°C) for the loop primers. The 3’ end stability of the primers: The 3’ end of the primers acts as the initiating point of the DNA polymerization and therefore, must be very stable and complementary with the target sequence. The following criteria are taken into consideration while selecting the primer sets. The 3’ ends of F2/B2, F3/B3 are designed so that the free energy is –4 kcal/ mol or less. LF/LB and the 5’ end of F1c/B1c are designed so that the free energy is –4 kcal/ mol or less. GC content: Primers are designed so that their GC content is about 40% to 65%. But, primers with GC content between 50% and 60% are selected as they are considered to give relatively better results. Secondary structures: Another important property of primers is the ability to form secondary structures or primer dimers. The primers are designed so that they do not form secondary structures. To avoid this condition, it is important to make sure that the 3’ end of the primer is non complementary. Example 2: Methodology: The throat swab samples are processed, and the RNA is extracted by using commercially available viral RNA extraction kit. (QIAamp Viral RNA Mini Kit; Catalogue number 52906). A commercially available kit is used for RT-LAMP; WarmStart® Colorimetric LAMP 2X Master Mix with UDG (Catalogue number M1804L). Primer mixture: A primer mixture is made prior to the actual RT-LAMP reaction. The primer mixture consists of 1.6 µM of FIP and BIP, 0.2 µM of F3 and B3, and 0.4 µM of LF/ LB (Table1). Table1: Concentration each primer to be incorporated in the primer mixture.

RT-LAMP reaction mixture: The assay was performed in a 20 µl reaction mixture containing 2 µl of 10 X primer mixture of 16 µM of both forward inner primer (FIP) and backward inner primer (BIP); 2 µM of both F3 and B3 primers and 4 µM of both loop forward (LF) and loop backward (LB).10 µl of WarmStart® Colorimetric LAMP 2X Master Mix with UDG (Catalogue number M1804L), 3.2 µl of nuclease free water, 0.8 µl of enhancer and 4 µl of RNA template. The reaction mixture was mixed thoroughly by vortex followed by a quick spin. Later the reaction was set at 65˚C for 30 minutes on a water bath. The results were observed with naked eye by looking at the color change. The reaction mixture for RT-LAMP consists of the following components mentioned in the table2. Table 2: The composition of RT-LAMP reaction

The reaction mixture is vortexed and spin down. It is ensured that the reaction solution has a bright pink color, which indicates initial high pH required for effective RT-LAMP reaction. The lids of the reaction tubes are closed and then the tubes are incubated at 65˚C for 30 minutes. Interpretation of results: The tubes are removed from the incubator and observed with the naked eye. The positive reaction is indicated by yellow colored reaction mix. On the other hand, the color of negative reaction remains pink. Testing of LAMP primers: Five LAMP primers human β-Actin gene were designed SEQ IDs 1-5. The sequences of LAMP primers of human β-Actin gene are listed (Table 3). Excellent result found housekeeping gene human β-actin gene at 65˚C for 30 minutes. Thus, β-Actin gene is used as internal control (IC) for further studies. The reaction color change from pink to yellow indicates positive reaction while no color change indicates negative reaction. The temperature of the IC tube reaction was set at 65˚C for 30 minutes (Fig.1). If reaction tubes exhibit orange colored reaction, incubate them for an additional 10 minutes at 65˚C. The β-Actin internal control primers designed in-house for RT-LAMP assay was found to be working efficiently as all the 34 samples showed positive results with RT-LAMP assay. Table 3. LAMP Primers for human β-Actin gene:

Validation of internal control RT-LAMP primers: A total of 34 clinical samples were tested by the two assays for validating the internal control primer beta actin. The 34 clinical samples taken for the study was processed at NIVMU COVID-19 testing facility. All the 34 samples gave the expected results when compared for the internal control primers designed in-house for RT-LAMP assay with the ICMR-NIV2019nCoV Assay Kit V 3.1. The results are given below (Fig.2 and Table 4). Table 4. Results of the parallel testing performed for comparison of internal control of RT-LAMP with ICMR-NIV2019nCoV Assay Kit V 3.1

Calculation of sensitivity and specificity: A total of 34 samples were tested using colorimetric RT-LAMP assay. The results were compared with real time RT-PCR. The comparative results were analyzed using the statistical analysis method to determine specificity and sensitivity. Sensitivity = Number of true positive / Number of true positive + Number of false negative Sensitivity = 100% Specificity = Number of true negatives / Number of true negatives + number of false positive Specificity = 100% Calculation of validity: The validity of test in comparison with real-time RT-PCR is calculated using a statistical analysis known as Cohen’s Kappa coefficient method (Cohen 1960). The Kappa value was calculated to be K = 1.0. It will thus be seen that the objects set forth above, among those made apparent from the preceding description, are efficiently attained, and since certain changes may be made in the constructions set forth without departing from the spirit and scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. The invention has been described with reference to preferred and alternate embodiments. Modifications and alterations will become apparent to those skilled in the art upon reading and understanding the detailed discussion of the invention provided herein. This invention is intended to include all such modifications and alterations insofar as they come within the scope of the present invention. These and other modifications of the preferred embodiments as well as other embodiments of the invention will be obvious from the disclosure herein, whereby the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation. Finally, to the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.

Claims

Claims: 1) Nucleotide sequences which target internal control (IC) gene (Beta actin), wherein said nucleotide sequences have SEQ ID NOs.1, 2, 3, 4 and 5. 2) The nucleotide sequence as claimed in claim 1, wherein said SEQ ID No.1 and SEQ ID No.2 are outer primer pairs; SEQ ID No.1 is forward outer primer β-Actin F3 and SEQ ID No.2 is backward outer primer β-Actin B3. 3) The nucleotide sequence as claimed in claim 1, wherein said SEQ ID No.3 and SEQ ID No. 4 are inner primer pairs; SEQ ID No.3 is forward inner primer β-Actin FIP and SEQ ID No.4 is backward inner primer β-Actin BIP. 4) The nucleotide sequence as claimed in claim 1, wherein said SEQ ID No.5 is forward loop primer β-Actin LF. 5) A primer mixture which targets internal control (IC) gene (Beta actin) based on loop mediated isothermal amplification (LAMP) technique comprising a sum total of 5 primers: (i) outer primer pairs comprising forward outer primer β-Actin F3 represented as SEQ ID NO: 1 and backward outer primer β-Actin B3 represented as SEQ ID NO: 2; (ii) inner primer pairs comprising forward inner primers β-Actin FIP represented as SEQ ID NO: 3 and backward inner primer β-Actin BIP represented as SEQ ID NO: 4; (iii) loop forward primer β-Actin LF represented as SEQ ID NO: 5; wherein the primers having GC content ranging from 40-65%, free energy up to –4 kcal/ mol or less at 3’ and 5’ ends of the primers and the primer melting temperature ranging from 59 - 61°C for forward and backward primers and 64 - 66°C for loop primers. 6) The primer mixture as claimed in claim 5, including three pairs of primers (F3 and B3): (FIP and BIP): (LF/LB) are at a molar ratio of (0.2: 1.6: 0.4) respectively. 7) A kit for detecting the internal control gene, wherein said kit comprising the primers mixture as claimed in claims 5-6. 8) A rapid loop mediated isothermal amplification method for detection of internal control (IC) gene in sample employed along with other RT-LAMP assays comprising the steps of: i) extracting RNA from a sample; ii) converting the RNA sample in complimentary DNA by using RT- LAMP technology and amplifying said DNA through LAMP amplification method involving the primer system comprising: iii) outer primer pairs: forward outer primers β-Actin F3 represented as SEQ ID NO: 1 and backward outer primer β- Actin B3: SEQ ID NO: 2; iv) inner primer pairs: forward inner primers β-Actin FIP represented as SEQ ID NO: 3 and backward inner primer β- Actin BIP represented as SEQ ID NO: 4 and loop forward primer β-Actin LF represented as SEQ ID NO: 5; v) detecting the amplified DNA by change of colour which is considered positive reaction when change of color is from pink to yellow. 9) The method as claimed in claim 8, wherein said method is conducted at 65˚C for 30 minutes.