WO2022013886A1

WO2022013886A1 - A system and a method for the diagnosis of an infectious disease

Info

Publication number: WO2022013886A1
Application number: PCT/IN2021/050677
Authority: WO
Inventors: Swarkar Sharma; Ekta Rai; Ramesh Sharma; Shakuntla SHARMA; Hemender SINGH
Original assignee: Swarkar Sharma
Priority date: 2020-07-11
Filing date: 2021-07-12
Publication date: 2022-01-20

Abstract

The present invention provides a system and a method for the diagnosis of an infectious disease by a novel nested loop-mediated isothermal amplification method with higher sensitivity and higher specificity. The system includes the designing of a plurality of primers based on the plurality of target regions of the target genome. The method employs a technique of reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP), loop-mediated isothermal amplification (LAMP) and nested loop-mediated isothermal amplification (LAMP). The present method provides the system and the method for the diagnosis of an infectious disease from a small microbial load, thereby allowing diagnosis at an early stage of infection.

Description

The Patents Act 1970 (39 of 1970)

&

The Patent Rules 2003 COMPLETE SPECIFICATION

(See Section 10 and rule 13) 1. TITLE OF THE INVENTION: A SYSTEM AND A METHOD FOR THE

DIAGNOSIS OF AN INFECTIOUS DISEASE

2. APPLICANT: a) Name: SWARKAR SHARMA b) Nationality: INDIA c) Address: 155, Ward No 5, Shankar Nagar, Jammu, Jammu and Kashmir, 185101, India

3. PREAMBLE OF THE DESCRIPTION: The following complete specification particularly describes the invention and the manner in which it is performed. FIELD OF THE INVENTION

The current invention relates to the field of molecular biology, it specifically relates to a system and a method for loop mediated nested nucleic acid isothermal amplification and the use thereof in nucleic acid (DNA or RNA) detection with high specificity and high sensitivity. The current invention can be used for the detection of microorganisms and more particularly can be used for the detection of microbes from a very small microbial load.

BACKGROUND OF THE INVENTION

Infectious diseases are caused by pathogenic microorganisms like bacteria, virus, parasites or fungi and can be spread directly or indirectly. An outbreak of the infectious disease leads to a pandemic, wherein a large population of individuals are affected over a wide geographical area, as can be seen in the present Covid-19 outbreak.

A sudden outbreak of a disease leaves a little or no time for assessing the situation, and therefore, early testing of the infectious disease is crucial in control of pandemics. Currently, amplification techniques like polymerase chain reaction (PCR) and its various types are used for the detection of the presence or absence of particular nucleic acid sequences. However, there is a limitation vis-a-vis inability to detect the presence or absence of target nucleic acid sequences present in low copy numbers in a sample.

Coronavirus (CoV) belongs to the Coronoaviridae family and is divided into three types: a (alpha), b (beta), g (gamma) and d (delta). Alpha and beta are only pathogenic to mammals and gamma mainly causes bird infections. CoV is mainly transmitted through direct contact with secretions or through aerosols and droplets. So far there are seven types of human coronavirus (HCoV) that cause human respiratory diseases: HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKUl, SARS-CoV, MERS-CoV and the novel coronavirus (2019). A very critical step for controlling the spread of infectious disesase, and curing them eary on is the identification and characetrization of the potential infected cases as well carriers so that can be quarantined and horizontal transmission of the infection is checked. However, all in use techniques and methods present high cost and limitations for Point-of-care (POC) diagnostics, which hampers its application for a large number of samples. This warrants development of assay that can be implimented at population level and with potential to screen bulk samples at the same time with very less viral load to detect potential positive cases in early stage.

At present, polymerise chain reaction (PCR) and antibody based testing are the dominant ways that global healthcare systems are using to test citizens for Covid-19. Both techniques have their caveats, and as the crisis unfolds alternative ways to screen for the deadly disease is critically required. Another technique used for the detection of covid-19 is antigen/antibody based assays. Antigen/ Antibody based assays detects the presence of the microbial proteins expressed by the microbe in a sample from a person or presence of an antibody against microbial antigen from the body of the infected person. In either of cases, if the target is present in sufficient concentration only it will bind to its specific antibody, resulting in detection. However, this technique gives results only when higher amount of target protein is present which is mostly absent in early stages of infection.

This makes the diagnosis of the microbe difficult during the early stages of infection, when the microbial load is less in an individual and the symptoms are not visible. In viral infections that are highly contagious, like recent outbreak of Covid-19, further, this leads to an increased risk of community spread, as the individual is himself not aware of his condition. Various other drawbacks pertaining to the current techniques include high false positives, sophisticated instruments for carrying out tests and therefore increased cost. Further, since the current techniques require sophisticated instruments and can only be carried out by trained technicians, there is a limitation for providing diagnosis at the point of care. However, each of these methodologies have their own limitations either these are cumbersome and require specific infrastructure or are unable to detect low viral load mainly in asymptomatic conditions critical for early detection or inability to be utilized for bulk population screening due to cost effectiveness or robustness with relatively high false positives or negatives. Presently, none of single methodology is in use that can solve all the issues in one go and the most critical of it viral detection in early phase of infection, critically needed at this juncture.

Loop-mediated isothermal amplification [LAMP] is a technique that relies on automaticcycles of the strand displacement DNA synthesis that is performed by a DNA polymerase with high strand displacement activity and a set of two specially designed inner and two outer primers. The best part about the methodology is requirement of very less specific resources. Studies have shown the importance of LAMP in the diagnosis of several viral diseases.

Exploiting the qualities of LAMP and adding innovative idea to include Nested Multiple Region detection simultaneously is going to make it a robust, easy to use, cheaper methodology and best part is potential to detect very low copy numbers of the virus, the case in early infectious stage of the disease making it difficult to be diagnosed.

In summary, the unique idea in present kit will help in increasing the capabilities of LAMP to detect viral load: 1. Bulk screening in population in cheaper way and to detect Viral copy number almost the double level of routine LAMP based assay.

2. Detection can be done by naked eye

3. Development of POC diagnostic kit which may not require specific equipment. The current invention discloses a LAMP based novel method for detecting any target polynucleotide, at very high sensitivity, high specificity and detection is faster because the target gets amplified faster with strand displacement isothermal PCR. This method can be used for POC (point of care) diagnostic tests, and samples can be collected by any of the currently known methods.

In view of the foregoing, there is a need to develop a system and a method for diagnosis of infectious disease in patients at the point of care during early stages of infection. There is also a need to develop a system and a method having high sensitivity and high specificity for the diagnosis of infectious disease in patients.

SUMMARY

One embodiment of the invention is an isothermal amplification method for detecting the presence of a target polynucleotide in a sample, wherein the target polynucleotide comprises a first target fragment called “A” and a second target fragment called “B”, wherein the first target fragment is 150-300 bp long and the second target fragment is 250-500 bp long and the first target fragment is nested in the 3’ portion of the second target fragment , the method comprising the steps of: a. Providing a set of eight primers, for simultaneously amplifying the first target fragment and the second target fragment and wherein the generated amplicons from the first target fragment and the second target fragment have the size range of 150- 500bp, wherein the primers are: i) A first forward primer called “Forward Inner Primer A (FIP-A)” comprised of two fragments F2 and Flc, wherein Flc fragment sequence is reverse complimentary to FI region on the first target fragment of the target polynucleotide and overhangs at 5 ’ end of F2, and wherein F2 hybridises in the forward direction to its complimentary sequence F2c on the first target fragment in the target polynucleotide and amplifies in 5’ to 3’ direction, and wherein the FI region is 40-60bp downstream region of F2c region on the first target fragment; ii) A second forward primer called F3 that hybridises to complimentary F3c region in the first target fragment of the target polynucleotide, wherein F3c is 0-60bp upstream to the F2c region in the first target fragment in the target polynucleotide; iii) A Third forward primer called Forward Loop Primer (FL), corresponding to single stranded FLc region in the first target fragment, that hybridises to a region in the loop formed due to self-complementarity of Flc overhang of FIP-A primer to FI region on the amplified strand displaced by extension of primer F3, wherein FL is located within 40-60bp loop region between FI and F2c regions in the in the first target fragment of the target polynucleotide; iv) A Fourth forward primer called “Forward Inner Primer B (FIP-B)” comprised of F5 and F4c fragments, wherein F4c is reverse complimentary to F4 region in the second target fragment of the target polynucleotide and overhangs at 5’ end of the F5 oligonucleotide, and wherein F5 hybridises in the forward direction to its complimentary sequence F5c on the second target fragment and amplifies it in 5’ to 3’ direction, and wherein the F4 region is 40-60bp downstream of the F5c region on the second target fragment,. v) A fifth forward primer F6 that hybridises to F6c region on the second target fragment in the target polynucleotide, wherein F6c is 0-60bp upstream to the F5c; vi) a first backward primer called “Backward Inner Primer (BIP)” , located in the overlapping region between the first and second target fragments, comprised of B2 and Blc fragments, wherein Blc is a reverse complimentary sequence to B1 region in the target polynucleotide, , and overhangs at the 5’ end of B2 oligonucleotide and further wherein B2 hybridises in the reverse direction to its complimentary sequence B2c in the target region in the target polynucleotide and amplifies it in the 5’ to 3’ direction, and wherein the B1 region corresponds to 40-60bp downstream region of B2c region on the target fragments. vii) A second Backward primer called B3 that hybridises to the B3c region of the target polynucleotide, wherein B3c region is 0-60bp upstream to the B2c region in the target polynucleotide. viii) A Third backward primer called Backward Loop Primer (BL) corresponding to single stranded BLc region in the target fragment, that hybridises to a region in the loop formed due to self-complementarity of Blc overhang of BIP primer to B 1 region on the amplified strand displaced by extension of primer B3 and wherein BL is located within the 40-60bp loop region between Blc and B2c regions in the target fragment of the target polynucleotide; b. adding the set of 8 primers from step (a) to a reagent mix comprising the target polynucleotide and an isothermal DNA polymerase and staring the amplification reaction for the target polynucleotide at temperature 60-70 degrees Celsius, wherein the reaction initiates simultaneously at from the FIP-B primer ,the F2 part of FIP-A, F5 part of FIP-B and B2 part of BIP followed by simultaneous extension and DNA synthesis, followed by displacement of amplicons formed by extension of FIP-A, FIP-B and BIP by F3, F6 and B3 primers, respectively; detecting the generated amplicons from the target polynucleotide sample.

In one embodiment, the method disclosed herein further comprises the step of reverse transcribing the target polynucleotide before adding the primers to it for the isothermal amplification reaction, if it the target polynucleotide is an RNA molecule.

In one embodiment, the method of claim 1 , wherein the 8 primers in the method disclosed herein have the GC content in range of 40-60%.

In one embodiment, the 8 primers are not complimentary sequences to each other, do not form secondary structures readily, do not form primer dimers and their Tm ranges from 55-65 degree Celsius. In one embodiment, the target polynucleotide in the method disclosed herein is a disease marker polynucleotide from an infectious microorganism.

In one embodiment, the nucleic acid disease marker comprises a nucleic acid marker for a disease caused by a microorganism selected from the group consisting of adenovirus B, adenovirus C, adenovirus E, Bordetella pertussis, mycobacterium tuberculosis (MTB), Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), Group A streptococcus, Group B streptococcus, Moraxella catarrhalis, Enterobacter aerogenes, Haemophilus parainfluenzae, Metapneumo Virus, Streptococcus pneumonia, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Coronavirus OC43, Coronavirus NL63, Coronavirus MERS, Coronavirus HKU1, Coronavirus 229E, Klibsiella pneumonia phoE, Klebsiella pneumonia KPC, Bocavirus type 2,4, and Bocavirus type 1,3.

In one embodiment, the infectious microorganism is a virus.

In one embodiment, the virus is a coronavirus.

In one embodiment, the coronavirus is the SARS-CoV2 virus.

In one embodiment, the target polynucleotide is the N gene from the SARS-CoV2 virus.

In one embodiment, the current invention encompasses a diagnostic kit comprising the set of primers for performing the method of claim 6 for detecting a disease marker polynucleotide.

In one embodiment, the method is a point-of service (POS) method performed at a POS location.

In one embodiment, the sequences of the primers comprise the nucleotide sequences with the following SEQ ID Nos (see Table 1): a. target fragment A comprises SEQ ID NO: 2; b. target fragment B comprises SEQ ID NO: 3; c. Flc comprises the sequence given in SEQ ID NO: 4; d. F2 comprises the sequence given in SEQ ID NO: 5; e. F3 comprises the sequence given in SEQ ID NO: 6; f. F4c comprises the sequence given in SEQ ID NO: 7; g. F5 comprises the sequence given in SEQ ID NO: 8; h. F6 comprises the sequence given in SEQ ID NO: 9; i. FL comprises the sequence given in SEQ ID NO: 10; j . B lc comprises the sequence given in SEQ ID NO: 11; k. B2 comprises the sequence given in SEQ ID NO: 12; l. B3 comprises the sequence given in SEQ ID NO: 13; and m. BL comprises the sequence given in SEQ ID NO: 14 BRIEF DESCRIPTION OF DRAWINGS

Figure 1 illustrates a nested LAMP reaction, disclosed herein;

Figure 2 shows the different regions of the SARS-Cov2 N gene, used for amplification using the primers and the method disclosed herein;

Figure 3 illustrates a flowchart showing the method for the diagnosis of an infectious disease, by the nested LAMP method disclosed herein;

Figure 4 shows gel photographs (the three pictures are replicates of the same experiment) for detection of nested LAMP reaction done in Example 1.

Lanes show; L-100 bp ladder; N-Negative; Ps- Stock Plasmid (with N gene); Db- Blood DNA; Pbet - Plasmid with 0.8M betaine; Ds- Saliva DNA; P(l.O)- Plasmid (dilution high to low).

To facilitate understanding, like reference numerals have been used, where possible, to designate like elements common to the figures. DETAILED DESCRIPTION

This section is intended to provide an explanation and description of various possible embodiments of the present invention. The examples used herein are intended only to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable the person skilled in the art to practice the embodiments used herein. Also, the examples/embodiments described herein should not be construed as limiting the scope of the embodiments herein.

As mentioned, there is a need of a system and a method for the diagnosis of an infectious disease at the point of care. Also, there is a need of the system and the method for the diagnosis of an infectious disease from a small microbial and small viral load, thereby enabling the diagnosis during the early stages of an infection.

As used herein, the terms “disease-causing agent”, “disease-causing organism” are used interchangeably to refer to viruses, bacteria, yeast, and other micro-organisms which may cause disease in a subject.

As used herein, the term “virus” refers to organisms which include nucleic acid message (either RNA or DNA) which allows their replication in infected host cells. The term virus includes both DNA viruses or RNA viruses.

As used herein, the term “micro-organism” refers to small unicellular or multicellular organisms which may infect cells, organs, tissues, or surfaces of plants or animals, including humans. The term microorganism includes bacteria (including mycoplasma), archea, fungus, yeast, parasites, and other small organisms.

As used herein, the term “bacteria” refers to small unicellular, prokaryotic organisms which may infect cells, organs, tissues, or surfaces of plants or animals, including humans. The term bacteria includes Gram negative bacteria and Gram positive bacteria.

As used herein, the phrase “nucleic acid markers indicative of’ or “disease markers” of a particular infection refers to nucleic acid molecules (including single-stranded and double-stranded DNA and RNA molecules) and fragments thereof, which are derived from disease-causing organisms, or are copies of, or are substantially similar to, or are complementary to, nucleic acid molecules derived from the organism which causes that particular infectious disease. Detection of nucleic acid markers indicative of a particular infection in a sample indicates that the disease-causing organism is, or was, present in the sample and thus that the subject has been exposed to the disease- causing organism, and likely suffers or suffered from the particular infection caused by that particular disease-causing organism.

As used herein, a “target” nucleic acid or molecule refers to a nucleic acid of interest. A target nucleic acid/molecule may be of any type, including single-stranded or double stranded DNA or RNA (e.g. mRNA).

As used herein, “complementary” sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. It is not necessary for every nucleotide base in two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other. In addition, sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other. For example, a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide.

The terms “point of service” (abbreviated POS) and “point of service system,” as used herein, refer to a location, and a system at that location, that is capable of providing a service (e.g. testing, monitoring, treatment, diagnosis, guidance, sample collection, verification of identity (ID verification), and other services) at or near the site or location of the subject. A service may be a medical service, and may be a non- medical service. In some situations, a POS system provides a service at a predetermined location, such as a subject's home, school, or work, or at a grocery store, a drug store, a community center, a clinic, a doctor's office, a hospital, etc. A POS system can include one or more point of service devices. In some embodiments, a POS system is a point of care system.

A “point of care” (abbreviated POC) is a location at which medical-related care (e.g. treatment, testing, monitoring, diagnosis, counseling, etc.) is provided. A POC may be, e.g. at a subject's home, work, or school, or at a grocery store, a community center, a drug store, a doctor's office, a clinic, a hospital, etc. A POC system is a system which may aid in, or may be used in, providing such medical-related care, and may be located at or near the site or location of the subject or the subject's health care provider (e.g. subject's home, work, or school, or at a grocery store, a community center, a drug store, a doctor's office, a clinic, a hospital, etc.).

In many cases, “point of care” location might be the same as the “point of service” location”.

Loop-mediated isothermal amplification (LAMP) is a method of DNA amplification that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands.

Embodiments:

One embodiment of the invention is an isothermal amplification method for detecting the presence of a target polynucleotide in a sample, wherein the target polynucleotide comprises a first target fragment called “A” and a second target fragment called “B”, wherein the first target fragment is 150-300 bp long and the second target fragment is 250-500 bp long and the first target fragment is nested in the 3’ portion of the second target fragment , the method comprising the steps of: a. Providing a set of eight primers, for simultaneously amplifying the first target fragment and the second target fragment and wherein the generated amplicons from the first target fragment and the second target fragment have the size range of 150-500bp, wherein the primers are: i) A first forward primer called “Forward Inner Primer A (FIP-A)” comprised of two fragments F2 and Flc, wherein Flc fragment sequence is reverse complimentary to FI region on the first target fragment of the target polynucleotide and overhangs at 5’ end of F2, and wherein F2 hybridises in the forward direction to its complimentary sequence F2c on the first target fragment in the target polynucleotide and amplifies in 5’ to 3’ direction, and wherein the FI region is 40-60bp downstream region of F2c region on the first target fragment; ii) A second forward primer called F3 that hybridises to complimentary F3c region in the first target fragment of the target polynucleotide, wherein F3c is 0-60bp upstream to the F2c region in the first target fragment in the target polynucleotide; iii) A Third forward primer called Forward Loop Primer (FL), corresponding to single stranded FLc region in the first target fragment, that hybridises to a region in the loop formed due to self-complementarity of Flc overhang of FIP-A primer to FI region on the amplified strand displaced by extension of primer F3, wherein FL is located within 40-60bp loop region between FI and F2c regions in the in the first target fragment of the target polynucleotide; iv) A Fourth forward primer called “Forward Inner Primer B (FIP-B)” comprised of F5 and F4c fragments, wherein F4c is reverse complimentary to F4 region in the second target fragment of the target polynucleotide and overhangs at 5’ end of the F5 oligonucleotide, and wherein F5 hybridises in the forward direction to its complimentary sequence F5c on the second target fragment and amplifies it in 5’ to 3’ direction, and wherein the F4 region is 40-60bp downstream of the F5c region on the second target fragment,. v) A fifth forward primer F6 that hybridises to F6c region on the second target fragment in the target polynucleotide, wherein F6c is 0-60bp upstream to the F5c; vi) a first backward primer called “Backward Inner Primer (BIP)” , located in the overlapping region between the first and second target fragments, comprised of B2 and B lc fragments, wherein B lc is a reverse complimentary sequence to B1 region in the target polynucleotide, , and overhangs at the 5’ end of B2 oligonucleotide and further wherein B2 hybridises in the reverse direction to its complimentary sequence B2c in the target region in the target polynucleotide and amplifies it in the 5’ to 3’ direction, and wherein the B1 region corresponds to 40-60bp downstream region of B2c region on the target fragments. vii) A second Backward primer called B3 that hybridises to the B3c region of the target polynucleotide, wherein B3c region is 0-60bp upstream to the B2c region in the target polynucleotide. viii) A Third backward primer called Backward Loop Primer (BL) corresponding to single stranded BLc region in the target fragment, that hybridises to a region in the loop formed due to self-complementarity of Blc overhang of BIP primer to B 1 region on the amplified strand displaced by extension of primer B3 and wherein BL is located within the 40-60bp loop region between Blc and B2c regions in the target fragment of the target polynucleotide; b. adding the set of 8 primers from step (a) to a reagent mix comprising the target polynucleotide and an isothermal DNA polymerase and staring the amplification reaction for the target polynucleotide at temperature 60-70 degrees Celsius, wherein the reaction initiates simultaneously at from the FIP-B primer ,the F2 part of FIP-A, F5 part of FIP-B and B2 part of BIP followed by simultaneous extension and DNA synthesis, followed by displacement of amplicons formed by extension of FIP-A, FIP-B and BIP by F3, F6 and B3 primers, respectively; detecting the generated amplicons from the target polynucleotide sample.

In one embodiment, after the displacement of amplicons formed by extension of FIP- A, FIP-B and BIP by F3, F6 and B3 primers, respectively, the amplicons that may be generated are, for example, FIP-A to BIP, FIP-B to BIP, F6 to BIP, F3 to BIP, FL to BIP, BL to FIP-A, BL to FIP-B.

In one embodiment, the 8 primers in the method disclosed herein have the GC content in range of 40-60%.

In one embodiment, the 8 primers are not complimentary sequences to each other, do not form secondary structures readily, do not form primer dimers and their Tm ranges from 55-65 degree Celsius.

In one embodiment, the target polynucleotide in the method disclosed herein is a disease marker polynucleotide from an infectious microorganism.

In one embodiment, the infectious microorganism is a virus. In one embodiment, the virus is a coronavirus.

In one embodiment, the coronavirus is the SARS-CoV2 virus.

In one embodiment, the current invention encompasses a diagnostic kit comprising the set of primers for performing the method disclosed herein for detecting a disease marker polynucleotide.

In one embodiment, the sequences of the primers comprise the nucleotide sequences with the following SEQ ID Nos (see Table 1): a. target fragment A comprises SEQ ID NO: 2; b. target fragment B comprises SEQ ID NO: 3; c. Flc comprises the sequence given in SEQ ID NO: 4; d. F2 comprises the sequence given in SEQ ID NO: 5; e. F3 comprises the sequence given in SEQ ID NO: 6; f. F4c comprises the sequence given in SEQ ID NO: 7; g. F5 comprises the sequence given in SEQ ID NO: 8; h. F6 comprises the sequence given in SEQ ID NO: 9; i. FL comprises the sequence given in SEQ ID NO: 10; j . B lc comprises the sequence given in SEQ ID NO: 11; k. B2 comprises the sequence given in SEQ ID NO: 12; l. B3 comprises the sequence given in SEQ ID NO: 13; and m. BL comprises the sequence given in SEQ ID NO: 14

In one embodiment, the method disclosed herein uses additional starting points for DNA synthesis. Additional amplification starting point means more locations where amplification can happen due to availability of more locations to be extended by polymerase in relation to normal LAMP in one reaction.

In one embodiment, the current method of detection of target nucleotides is much faster and the nucleotide can be detected in 30 mins to one hour.

In one embodiment, less than 1 picogram of DNA can be detected by the current method. In one embodiment, 10 or less than 10 molecules of target polynucleotide can be detected by the method disclosed herein.

The present embodiment provides the system for diagnosing the infectious disease by employing a technique of Loop-mediated isothermal amplification. In an embodiment, the system for diagnosing viral load of SARS-CoV-2 in covid-19 patients is provided. The system includes the designing of primers based on the plurality of target regions of the SARS-CoV-2 virus.

Since, the SARS-CoV-2 virus isolated from the infected patients has a single stranded RNA genome; the present embodiment employs the techniques of reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) and loop-mediated isothermal amplification (LAMP) for the diagnosis of the covid-19. In an embodiment, two or more LAMP amplifications are carried out simultaneously in the target genome. In an embodiment, the multiple LAMP amplifications are nested LAMP amplifications (shown in figure 3). In an embodiment, the nested LAMP amplifications allow the diagnosis of very small microbial load. At step 402, a sample is collected from an infected individual. In an embodiment, the sample is a body fluid. In an embodiment, the sample is collected by swabbing a nasal, oral and respiratory part of an infected individual.

At step 404, a viral RNA is isolated from the sample collected at step 402. In an embodiment, the viral RNA is isolated by using a kit already available in the market such as, but not limited to, QIAamp Viral RNA mini kit, Ambion’s MagMAX™ viral RNA isolation kit and NucleoSpin RNA virus kit.

At step 406, a complementary DNA strand is synthesised for a single stranded viral RNA genome.

At step 408, the single stranded DNA synthesised in step 406 is amplified through the process of loop-mediated isothermal amplification technique (LAMP). In an embodiment, the amplification requires a temperature conditions ranging between 60- 65 °C. In an embodiment, the LAMP requires time duration of 30 mins to an hour for the amplification. The process of loop-mediated isothermal amplification (LAMP) includes the following steps:

The released complementary DNA strand from step 406 serves as a template for the loop-mediated isothermal amplification. In an embodiment, the released complementary DNA strand is dumbbell shaped. In an embodiment, the 3’ end and the 5 ’end of the complementary DNA strand folds and anneal to form the dumbbell-shaped structure.

In an embodiment, two or more LAMP amplifications are carried out simultaneously in the target genome. In an embodiment, the multiple LAMP amplifications are nested LAMP amplifications (shown in figure 3). In an embodiment, the nested LAMP amplifications allow the diagnosis of a small viral load.

Hybridization conditions may typically include salt concentrations of less than about 1M, more usually less than about 500 mM, for example less than about 200 mM. Hybridization temperatures is typically greater than about 50° C but within range of 50-70°C. Longer fragments may require higher hybridization temperatures for specific hybridization as is known in the art.

Any of the known detection methods can be used for detecting the product obtained from the current LAMP method disclosed herein. Examples of these methods include, but are not limited to, include turbidity, agarose gel electrophoresis, fluorescence based assays, colorimetric detection using naked eyes and detection using UV light. Real-time monitoring based on turbidity using real-time turbidity metre (e.g.Loopamp Realtime Turbidimeter LA-200, LA-320, LA-500, Eiken Chemical Co., Ltd., Tokyo, Japan) is commonly used for LAMP product detection. LAMP assay is also monitored for the white precipitation caused by the presence of magnesium pyrophosphate, Mg2P2C>7 (which may be a by-product of LAMP reaction) at optical density 650 nm every 6 s.

While the disclosure has been presented with respect to certain specific embodiments, it will be appreciated that many modifications and changes may be made by those skilled in the art without departing from the spirit and scope of the disclosure. It is intended, therefore, by the appended claims to cover all such modifications and changes as fall within the true spirit and scope of the disclosure.

Examples

Example 1: Primer Designing and preparation of dilutions Primers (oligonucleotides) were synthesized for targeting specific region of the nCOV-2 genome (NC_..045512.2) [table 1] specific for the specific conserved region of the nCOV-2 genome in N gene Gene ID: 43740575, NC_045512.2 (28274-29533 bp). For experimental work partial region was artificially synthesized (SEQ ID NO: 1) and cloned from 28501-29290 bp) in Plasmid (pUCminus MCS) (commercially) from Eurofin Genomics India Pvt Ltd. The plasmid was used as a positive control sample and all the reaction conditions were standardized using the Plasmid. Oligos were designed and got synthesized through Eurofin genomics pvt ltd. 100 pL of primers stocks were made in nuclease free water. lOpM of primer dilution (100 pL) aliquots were prepared for all the three sets of the primer. Table 1

LAMP PCR reaction mix

The LAMP PCR reaction was carried out in 10- 15m L PCR tube consisting of NEB Isothermal amplification buffer, NEB MgSCL, NEB Bst 2.0/3.0, primers, nuclease free water. The components are added in various proportions in the reaction and generalized versions are mentioned in table 1 and table 2 One microliter of each non-template control (NTC), positive control plasmid and positive and negative RNA samples were used as samples in the reaction.

Standardization of reaction conditions

To determine the optimum temperature for the LAMP PCR reaction. Gradient PCR was run for the three sets of the primer individually taking NTC, positive control and positive RNA sample. The gradient was run between the temperature range of 50-70 °C in two phases in the first phase the gradient was run for 50-61 °C range and in the second phase the PCR was carried out in the temperature range of 60-70 °C.

To determine the sensitivity of our reaction different fold of dilutions and replicates (4, 8, 16 and 32 fold) of the positive sample was prepared and LAMP was carried out respectively.

LAMP assay

Table 2 : default set Components for Nested LAMP (Dual Alternate LAMP) used for 10-15 pL of reaction volume

Gel electrophoresis

To visualize the LAMP results, 3% of 50 mL agarose gel was prepared in IX TAE buffer, 1.5pL of EtBr was added into the gel to visualize the results. 3pL of ready to use lOObp DNA ladder (BR Biochem, Cat No: BM001-R500) was used as a marker. 2 m L of loading dye (bromophenol blue) and 5 pL the LAMP amplicons were loaded in each well. The gel was run at the 70 volts for 45 to 60 minutes. The gel was then visualized under UV trans illuminator (GeNei ™). Figure 4 shows the results of these assays . The lanes Ps and P1-P10 shows samples with different dilutions.

Table 3

Results After performing the gradient LAMP it was observed that many primer combinations did not show specific amplification at any of the temperature in the range of 50-70°C and various concentrations were needed to adjust to achieve amplification. Finally primer set combinations and concentrations were devised to be showing specific amplification and standardized to work at the optimum temperature of 68 °C for one hour.

It was observed that there was no significant change in the LAMP specificity after adding 0.8M Betaine, therefore it was not used for further experiments. Different dilutions of the samples were made so as to determine the sensitivity of the reaction and it was found that our LAMP PCR was very sensitive and can detect at 2⁵ dilutions (1/32 times dilution of original sample). Amplification of all these samples were confirmed through agarose gel electrophoresis showing very bright LAMP pattern in both positive control and positive sample and no amplification in the negative controls.

Claims

I/We Claim:

1. An isothermal amplification method for detecting the presence of a target polynucleotide in a sample, wherein the target polynucleotide comprises a first target fragment called “A” and a second target fragment called “B”, wherein the first target fragment is 150-300 bp long and the second target fragment is 250-500 bp long and the first target fragment is nested in the 3’ portion of the second target fragment , the method comprising the steps of: a. Providing a set of eight primers, for simultaneously amplifying the first target fragment and the second target fragment and wherein the generated amplicons from the first target fragment and the second target fragment have the size range of 150-500bp, wherein the primers are: i. A first forward primer called “Forward Inner Primer A (FIP-A)” comprised of two fragments F2 and Flc, wherein Flc fragment sequence is reverse complimentary to FI region on the first target fragment of the target polynucleotide and overhangs at 5’ end of F2, and wherein F2 hybridises in the forward direction to its complimentary sequence F2c on the first target fragment in the target polynucleotide and amplifies in 5’ to 3’ direction, and wherein the FI region is 40-60bp downstream region of F2c region on the first target fragment; ii. A second forward primer called F3 that hybridises to complimentary F3c region in the first target fragment of the target polynucleotide, wherein F3c is 0-60bp upstream to the F2c region in the first target fragment in the target polynucleotide; iii. A Third forward primer called Forward Loop Primer (FL), corresponding to single stranded FLc region in the first target fragment, that hybridises to a region in the loop formed due to self complementarity of Flc overhang of FIP-A primer to FI region on the amplified strand displaced by extension of primer F3, wherein FL is located within 40-60bp loop region between FI and F2c regions in the in the first target fragment of the target polynucleotide; iv. A Fourth forward primer called “Forward Inner Primer B (FIP-B)” comprised of F5 and F4c fragments, wherein F4c is reverse complimentary to F4 region in the second target fragment of the target polynucleotide and overhangs at 5’ end of the F5 oligonucleotide, and wherein F5 hybridises in the forward direction to its complimentary sequence F5c on the second target fragment and amplifies it in 5’ to 3’ direction, and wherein the F4 region is 40-60bp downstream of the F5c region on the second target fragment,. v. A fifth forward primer F6 that hybridises to F6c region on the second target fragment in the target polynucleotide, wherein F6c is 0-60bp upstream to the F5c; vi. a first backward primer called “Backward Inner Primer (BIP)” , located in the overlapping region between the first and second target fragments, comprised of B2 and Blc fragments, wherein Blc is a reverse complimentary sequence to B 1 region in the target polynucleotide, , and overhangs at the 5’ end of B2 oligonucleotide and further wherein B2 hybridises in the reverse direction to its complimentary sequence B2c in the target region in the target polynucleotide and amplifies it in the 5’ to 3’ direction, and wherein the B 1 region corresponds to 40-60bp downstream region of B2c region on the target fragments. vii. A second Backward primer called B3 that hybridises to the B3c region of the target polynucleotide, wherein B3c region is 0-60bp upstream to the B2c region in the target polynucleotide. viii. A Third backward primer called Backward Loop Primer (BL) corresponding to single stranded BLc region in the target fragment, that hybridises to a region in the loop formed due to self complementarity of Blc overhang of BIP primer to B1 region on the amplified strand displaced by extension of primer B3 and wherein BL is located within the 40-60bp loop region between B lc and B2c regions in the target fragment of the target polynucleotide; b. adding the set of 8 primers from step (a) to a reagent mix comprising the target polynucleotide and an isothermal DNA polymerase and staring the amplification reaction for the target polynucleotide at temperature 60-70 degrees Celsius, wherein the reaction initiates simultaneously at from the FIP-B primer, ,the F2 part of FIP-A, F5 part of FIP-B and B2 part of BIP followed by simultaneous extension and DNA synthesis, followed by displacement of amplicons formed by extension of FIP-A, FIP-B and BIP by F3, F6 and B3 primers, respectively; c. detecting the generated amplicons from the target polynucleotide sample.

2. The method of claim 1, wherein it further comprises the step of reverse transcribing the target polynucleotide in step (b) if it the target polynucleotide is an RNA molecule.

3. The method of claim 1, wherein the sequences of the primers comprise the nucleotide sequences with the following SEQ ID Nos: target fragment A comprises SEQ ID NO: 2; target fragment B comprises SEQ ID NO: 3;

Flc comprises the sequence given in SEQ ID NO: 4; F2 comprises the sequence given in SEQ ID NO: 5;

F3 comprises the sequence given in SEQ ID NO: 6;

F4c comprises the sequence given in SEQ ID NO: 7;

F5 comprises the sequence given in SEQ ID NO: 8;

F6 comprises the sequence given in SEQ ID NO: 9;

FL comprises the sequence given in SEQ ID NO: 10;

Blc comprises the sequence given in SEQ ID NO: 11;

B2 comprises the sequence given in SEQ ID NO: 12;

B3 comprises the sequence given in SEQ ID NO: 13; and

BL comprises the sequence given in SEQ ID NO: 14

4. The method of claim 1, wherein the 8 primers have GC content in range of 40-60%.

5. The method of claim 1, wherein the 8 primers are not complimentary sequences to each other, do not form secondary structures readily, do not form primer dimers and their Tm ranges from 55-65 degree Celsius.

6. The method of claim 1, wherein the target polynucleotide is a disease marker polynucleotide from an infectious microorganism.

7. The method of claim 6, wherein the nucleic acid disease marker comprises a nucleic acid marker for a disease caused by a microorganism selected from the group consisting of adenovirus B, adenovirus C, adenovirus E, Bordetella pertussis, mycobacterium tuberculosis (MTB), Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), Group A streptococcus, Group B streptococcus, Moraxella catarrhalis, Enterobacter aerogenes, Haemophilus parainfluenzae , Metapneumo Virus, Streptococcus pneumonia, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Coronavirus OC43, Coronavirus NL63, Coronavirus MERS, Coronavirus HKU 1 , Coronavirus 229E, Klibsiella pneumonia phoE, Klebsiella pneumonia KPC, Bocavirus type 2,4, and Bocavirus type 1,3.

8. The method of claim 7, wherein the infectious microorganism is a virus.

9. The method of claim 8, wherein the virus is a coronavirus.

10. The method of claim 9, wherein the coronavirus is the SARS-CoV2 virus.

11. The method of claim 1 , wherein the target polynucleotide is the N gene from the SARS-CoV2 virus.

12. A diagnostic kit comprising the set of primers for performing the method of claim 6 for detecting a disease marker polynucleotide.

13. The method of claim 1, wherein the method is a point-of service (POS) method performed at a POS location. Dated this 12^th Day of July, 2021

Signature of Patent Agent:

(RahulBagga)

IN/PA-2366