Primer panel for multiplex PCR-based detection of drug-resistant plasmodium, and kit comprising the same

Abstract

The present disclosure relates to a multiplex PCR-based method for antimalarial resistance surveillance. In particular, the present disclosure provides a Plasmodium falciparum Multi Drug Resistance 15 panel (PfMDR15) to detect mutations in drug-resistance genes of P. falciparum from the given sample, and a kit comprising the same. The present invention also provides a rapid, sensitive, multiplex polymerase chain reaction (MPCR) employing said primer panel for detecting mutations in drug-resistance genes of P. falciparum and its variants. This is needed for carrying out surveillance studies to track P. falciparum variants and correlate with drug efficacy.

Description

[0001] PT / 2026 / 11086

[0002] PRIMER PANEL FOR MULTIPLEX PCR-BASED DETECTION OF DRUG-RESISTANT PLASMODIUM, AND KIT COMPRISING THE SAME

[0003] TECHNICAL FIELD OF THE INVENTION

[0004] The present disclosure relates to a primer panel, multiplex PCR-based method and composition for the amplification or detection of mutations in drug-resistance genes of Plasmodium falciparum, and a kit comprising the same.

[0005] BACKGROUND OF THE INVENTION

[0006] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the present invention, or that any publication specifically or implicitly referenced is prior art.

[0007] Malaria is a serious disease caused by Plasmodium parasites, with Plasmodium falciparum fP. falciparum) being the most dangerous type. This disease is a huge problem in over 100 countries, mainly in tropical and subtropical regions, and it hits poorer countries the hardest. The main way to treat malaria is with antimalarial drugs, but these drugs are becoming less effective because the parasites are developing resistance to them. This resistance happens because the drugs are not used optimally, and their effects don't last long enough.

[0008] Resistance mutations in parasites also pose a significant threat to malaria elimination. Even though molecular surveillance of some resistance markers has been done in the Indian population, there are no reports on the current status of drug-resistance in the parasites in sub-continent. With the emergence of ACT resistance in south-east Asia, it is imperative that regular molecular surveillance be done in malaria endemic regions of India. Mutations in genes DHFR, DHPS, CRT, K13, and MDR1 are known to be associated with resistance of parasites to antimalarials.

[0009] As a result, resistant strains of P. falciparum are spreading quickly. For example, chloroquine resistance is now common in Central and South America, Africa, and Southeast Asia. Sulfadoxine-pyrimethamine is no longer effective in many parts of Africa, Southeast and South Asia, South America, and Oceania. Even artemisinin, the most effective drug we have, is starting to lose its effectiveness in Southeast Asia. Developing new antimalarial drugs is very important but also very slow. Artemisinin, for instance, was discovered in 1972 but only became widely used in the 1990s. To fight drug-resistance, we need to understand how widespread it is, and which drugs the parasites are resistant to. This involves studying the parasites' genes to find mutations that causePT / 2026 / 11086

[0010] resistance. Whole genome sequencing (WGS) is one way to do this, but it is very expensive and not practical for large-scale use.

[0011] Understanding and fighting drug-resistance in malaria is crucial for global health. Advances in genomic sequencing, especially targeted amplicon sequencing, provide promising and affordable ways to monitor drug-resistance in P. falciparum. This ensures that we can continue to control and treat malaria effectively.

[0012] Genetic sequence determines the structure of the protein, and the structure determines the function. Therefore, advantages of identifying the genetic variations in proteins, which are targets of antimalarial drugs or involved in developing resistance, are multifold. The parasite genetic information will help in deciding if the treatment / drugs prescribed in a region need to be changed or not. It will also help in determining how closely related the parasitic population in a region is to other populations. This might help in identifying whether malaria infections are being imported into the region due to migratory population. Therefore, investigating the parasite genetic polymorphisms will substantially help in eliminating malaria.

[0013] Determining the prevalence of these mutations in malaria endemic regions in India will help the national malaria programme in suggesting best treatment regimens, specific to each region. The genetic sequence of parasites from these different regions will also help in understanding the evolution of parasites and their point of origin.

[0014] A more affordable option is targeted amplicon sequencing, which focuses on specific parts of the genome known to be linked to drug-resistance. This method is cheaper because it allows many samples to be processed at once, increasing efficiency. Some studies, like "Drug-resistance and Vaccine Target Surveillance of P. falciparum Using Nanopore Sequencing in Ghana" and "Flexible and Cost-Effective Genomic Surveillance of P. falciparum Malaria with Targeted Nanopore Sequencing," have developed methods to do this, but they only cover up to six genes and target known mutations.

[0015] Thus, conventional methods such as comprehensive genome sequencing are expensive and impractical for large-scale surveillance. Targeted amplicon sequencing, focusing on specific drugresistance genes, offers a cost-effective alternative. However, existing panels cover only a limited number of genes and mutations. There is a need for a primer panel or a method, which can target more genes and is cost-effective and scalable.PT / 2026 / 11086

[0016] OBJECTS OF THE INVENTION

[0017] An object of the present invention is to develop a feasible, robust and easy-to-implement surveillance method for the detection of mutations in drug-resistance genes of drug-resistant P. falciparum using highly sensitive multiplex nested PCR technique

[0018] An object of the present invention is to provide a composition and methods that can more effectively detect mutations in drug-resistance genes of drug-resistant P. falciparum from samples.

[0019] An object of the present invention is to provide a primer panel comprising a comprehensive set of unique primers with an optimized method for performing multiplex PCR to detect mutations in drug-resistance genes of drug-resistant P. falciparum in the samples

[0020] Another object of the present invention is to provide a kit for detecting and quantifying mutations in drug-resistance genes of drug-resistant P. falciparum and its variants in the samples.

[0021] SUMMARY OF THE INVENTION

[0022] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0023] The present invention relates to a primer panel for amplification or detecting mutations in drug resistant genes of Plasmodium falciparum said primer panel having pools of primers. Each pool can comprise:

[0024] (i) a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof; and

[0025] (ii) a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof.

[0026] The set of primers can correspond to one or more drug resistant genes having SEQ ID Nos:79-93.PT / 2026 / 11086

[0027] The present invention provides a composition for amplification or detecting mutations in drug resistant genes of Plasmodium falciparum. The composition can comprise the primer panel of the present invention and optionally a master mix containing dNTPs, buffer, and a DNA polymerase.

[0028] The present invention provides a method for the whole genome amplification or detection of drugresistant genes for Plasmodium falciparum in a sample by multiplex polymerase chain reaction (PCR), wherein the method comprises:

[0029] (i) mixing half of the sample containing the target DNA with a primer pool containing a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof or variants thereof, a master mix comprising deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to obtain the PCR product;

[0030] (ii) mixing the other half of the sample containing the target DNA with a primer pool containing a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof, a master mix comprising deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to obtain the PCR product; and

[0031] (iii) processing the PCR products thus obtained for library preparation for sequencing.

[0032] The multiplex PCR can comprise the steps of:

[0033] (i) Initial denaturation at 95°C for 1 minute for 1 cycle and

[0034] (ii) Denaturation at 95°C, annealing and extension at 63 °C for 35 cycles and holding at 4°C indefinitely.

[0035] The amplicons in each of the PCR products can have about 1.5 kb size.

[0036] The present invention relates to an assay for detecting mutations in drug-resistance genes of Plasmodium falciparum. The assay can comprise the steps of:

[0037] a) mixing a sample with a primer panel having pools of primers, each pool comprising:PT / 2026 / 11086

[0038] a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof; and

[0039] a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof.

[0040] b) performing multiplex PCR to amplify target regions of drug-resistance genes having SEQ ID Nos:79-93;

[0041] c) sequencing the amplified products; and

[0042] d) analyzing the sequence data to detect mutations in said drug-resistance genes.

[0043] The present invention provides a method for the antimalarial surveillance of P. falciparum in a sample. The method can comprise the step of:

[0044] a) contacting a sample with a primer panel having pools of primers, each pool comprising:

[0045] a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof; and

[0046] a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof.

[0047] b) performing multiplex PCR to amplify target regions of drug-resistance genes having SEQ ID Nos:79-93;

[0048] c) sequencing the amplified products.

[0049] The sample can be blood sample, EDTA Blood or Dried blood spot (DBS).

[0050] The present invention provides a kit for amplification or detecting mutations in drug resistant genes of Plasmodium falciparum comprising:PT / 2026 / 11086

[0051] (i) a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof; and

[0052] (ii) a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof.

[0053] The kit can further comprise deoxynucleotide triphosphates (dNTPs), a buffer, DNA polymerase, optionally, a positive control containing synthetic DNA segments of amplification targets, optionally a negative control containing a reaction mixture without any nucleic acid; and nuclease-free water.

[0054] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

[0055] BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The invention has other advantages and features, which will be more readily apparent from the following detailed description of the invention and the appended claims, when taken in conjunction with the accompanying drawings, in which:

[0057] Figure 1 displays individual amplicons produced under uniform PCR conditions. Wells 1 and 2: DHFR-TS amplicons, Wells 3, 4, and 5: CRT amplicons, Well 6: CYTB amplicons. Wells 7 to 12: MRP1 gene amplicons, Wells 12 and 13: kl3 gene amplicons. Wells 14 to 17: ATP6 gene amplicons. Wells 18 to 20: MRD1 amplicons, Wells 21 and 22: TCTP gene amplicons. Wells 23 to 25: PPPK-DHPS gene amplicons, Wells 26 and 27: DHODH amplicons. Wells 28 to 30: MDR1 amplicons Wells 31 to 33: MDR2 amplicons. Well 34 shows the Fd amplicon, and wells 35 to 37: Exo. Wells 38 and 39: ARPS 10 amplicons.

[0058] Figure 2 depicts A) the gel image of amplicon pools A and B; and B) the sequencing coverage for each gene, differentiated by varying shades.

[0059] Figure 3 depicts A) the gel image showcases the specificity of the PCR reactions. In wells 1 and 2, the MDR panel is used in conjunction with P. falciparum genomic DNA. Wells 3 and 4 display results from PCR with the MDR panel and host genomic DNA as the template. Wells 5 and 6 illustrate the PCR outcomes utilizing human genomic DNA with specific primers meant for aPT / 2026 / 11086

[0060] positive control; and B) PCR sensitivity across a gradient of parasitaemia levels, decreasing from 4%, 2%, 1%, 0.5%, 0.1%, 0.05%, to 0.01%, sequentially arranged from well 1 to well 7.

[0061] Figure 4 illustrates the gene coverage at varying levels of parasitemia. Panel A) shows coverage at 4% parasitemia, Panel B) depicts coverage at 0.05% parasitemia, and Panel C) presents coverage at 0.01% parasitemia.

[0062] Figure 5 shows the brief process steps covered in Multiplex PCR Protocol and Oxford Nanopore Technology (ONT) sequencing.

[0063] DETAILED DESCRIPTION OF THE INVENTION:

[0064] While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.

[0065] Throughout the specification and claims, the following terms take the meanings explicitly associated herein unless the context clearly dictates otherwise. The meaning of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on." Referring to the drawings, like numbers indicate like parts throughout the views. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.

[0066] The tables, figures and protocols have been represented where appropriate by conventional representations in the drawings, showing only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

[0067] As used herein, a sample refers to any substance containing or presumed to contain nucleic acid and includes a sample isolated from human.

[0068] As used herein, the terms “nucleic acid”, “polynucleotide” and “oligonucleotide” refer to primers, oligomer fragments to be detected, and oligomer controls.

[0069] As used herein, the term “Primer” refers to an oligonucleotide, whether occurring naturally or produced synthetically, which is capable of acting as a point of initiation of synthesis when placedPT / 2026 / 11086

[0070] under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced. The exact lengths of the primers will depend on many factors, including temperature, source of primer and use of the method.

[0071] As used herein, the term “amplification” refers to methods to increase the number of copies of a nucleic acid molecule. The resulting amplification products are typically called “amplicons.” Amplification of a nucleic acid molecule (such as a DNA or RNA molecule) refers to use of a technique that increases the number of copies of a nucleic acid molecule (including fragments). In some examples, an amplicon is a part nucleic acid from a cell, or acellular system, such as mRNA or DNA that has been amplified.

[0072] As used herein, the term “nucleoside triphosphate” is used herein to refer to nucleosides present in either DNA or RNA and thus includes nucleosides, which incorporate adenine, cytosine, guanine, thymine and uracil as base, the sugar moiety being deoxyribose or ribose

[0073] Embodiments of the present disclosure relates to a multiplex PCR-based method for disease surveillance. In particular, the present disclosure provides a multiplex primer panel, which can be designated as PfMDR15 for illustration purpose to detect mutations in drug-resistance genes of P. falciparum from the given sample, and a kit comprising the same. The present invention also provides a rapid, sensitive, and improved multiplex polymerase chain reaction (MPCR) employing said primer panel for detecting mutations in drug-resistance genes of P. falciparum and its variants. This is needed for carrying out large-scale screening and surveillance studies to track P. falciparum variants and correlate with drug efficacy.

[0074] In some embodiments of the present invention, the methods and compositions described herein may be used for detecting mutations in drug-resistance genes of P. falciparum and its variants, in samples. Said method may comprise providing a sample with reagents for detection, incubating the sample under suitable conditions to allow binding of the primers to nucleic acid corresponding to one or more drug-resistance genes (Table 1) present in the P. falciparum. In some embodiments, the mutations are selected from but not limited to synonymous, non- synonymous, Indel and frame shift mutations.

[0075] Table 1: Drug-resistance genes

[0076]

[0077] PT / 2026 / 11086

[0078]

[0079] In some embodiments of the present invention, the methods and compositions described herein may be used for detecting mutations in target molecules, wherein the nucleic acid comprises a target nucleotide sequence that may be used to distinguish among drug-resistant species / strains from one another.PT / 2026 / 11086

[0080] In an embodiment, the primer set of the present invention amplifies shorter 1.5 kb genomic amplicons in size between adjacent PCR fragments with uniform efficiency, covering 15 drugresistance genes of the P. falciparum genome (Table 1).

[0081] In an embodiment, the present disclosure provides a PfMDR15 primer panel having the nucleotide sequence of SEQ ID NO: 1-78 for detecting mutations in drug-resistance genes of P. falciparum and its variants found in clinical samples.

[0082] In an embodiment, the present disclosure provides a PfMDR15 primer panel having the nucleotide sequence of SEQ ID NO: 1-78 for amplification of drug-resistance genes of P. falciparum and its variants found in clinical samples.

[0083] In an embodiment, the present invention relates to a primer panel for amplification or detecting mutations in drug resistant genes of Plasmodium falciparum. The primer panel can have pools of primers, each pool can comprise:

[0084] (iii) a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof; and

[0085] (iv) a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof.

[0086] For illustration purpose, the primer panels may be partitioned in pool of primers as follows:

[0087] (i) a set of pool A primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, and 78 specific for drug-resistance genes in drug-resistant P. falciparum genome; and

[0088] (ii) a set of Pool B primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, and 76 specific for drug-resistance genes in drug-resistant P. falciparum genome.

[0089] In an embodiment, the present invention relates to a PfMDR15 primer panel having the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,PT / 2026 / 11086

[0090] 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77 as forward primers (Table 2 provided in Examples).

[0091] In an embodiment, the present invention relates to a PfMDR15 primer panel having the nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78 as reverse primers (Table 2).

[0092] In an embodiment, the primer panel of the present invention can target the complete sequences of one or more drug-resistance genes of P. falciparum, including DHFR-TS, PPPK-DHPS, CYTB, DHODH, ATP6, TCTP, MRP1, MRD1, MDR1, MDR2, K13, CRT, ARPS 10, Fd, and Exo having SEQ ID NOs:79-93 respectively. The panel can be divided into two pools such that adjacent amplicons are assigned to different pools, preventing the formation of excessively long PCR products and ensuring robust amplification of overlapping fragments. For genes exceeding 1.5 kb, the amplicons can be split into multiple fragments with overlapping regions. The PCR products may be pooled and sequenced using high-throughput platforms such as Oxford Nanopore Technology (ONT) or any other technology known to a person skilled in the art, enabling rapid, high-coverage amplification and sequencing. This facilitates surveillance and monitoring of variants, and epidemiological studies.

[0093] In an embodiment, the present invention provides a composition comprising the primer panel as disclosed herein, and optionally a master mix of deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase.

[0094] The buffer may be 2x reaction buffer containing optimized concentrations of MgC12, dNTP’s, HiFi polymerase, hot start antibodies and ToughMix chemistry.

[0095] The DNA polymerase may be selected from but not limited to Quantabio repliQa HiFi ToughMix, Takara PrimeSTAR GXL, NEB Q5 Hot Start High-Fidelity 2X Master Mix, and NEB LongAmp Hot Start Taq DNA Polymerase.

[0096] In an embodiment, the present invention can provide a method for whole genome amplification or detection of drug-resistant genes for Plasmodium falciparum in a sample by multiplex PCR. The method can comprise dividing the sample into two halves, each mixed with-PT / 2026 / 11086

[0097] (i) a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof; and

[0098] (ii) a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof along with PCR reagents such as a master mix comprising deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to obtain the PCR products. The DNA can be amplified in two separate reactions. The PCR products can be pooled and processed for library preparation and sequencing. The sequence may be performed in such a manner that each PCR product amplicon can be approximately 1.5 kb in size, with overlapping regions of about 0.5 kb. The rationale behind splitting into two pools can be that the forward primer of the second amplicon can pair with the reverse primer of the first amplicon, generating smaller overlapping amplicons. This design prevents the formation of excessively long PCR products, as shorter amplicons are preferentially amplified, ensuring robust and efficient amplification. This design may ensure complete genome coverage and may facilitate accurate assembly of the genome from sequencing data. The method of amplification can be targeted amplification.

[0099] The samples from infected persons may be processed using this method, and the resulting sequencing data may be used to identify the variants and track outbreaks.

[0100] The multiplex PCR can comprise:

[0101] (ii) denaturation at 90-99 °C for 20-40 seconds, followed by one or more cycles of 90-99 °C denaturation for 10-40 seconds, preferably, at 95 °C; and

[0102] (ii) annealing and extension at 40-70 °C, preferably 63 °C and holding at 4°C indefinitely.

[0103] The method may be performed using a blood sample, EDTA Blood or Dried Blood Spot.

[0104] In an embodiment, optionally the PCR products can be analysed by agarose gel electrophoresis. This step may confirm that the pool A and pool B PCRs have worked for each sample before sequencing and ensure that the PCR product is devoid of primer dimers (this can affect sequence data quality).PT / 2026 / 11086

[0105] The reagents can be Invitrogen™ UltraPure™ Agaroselnvitrogen- Thermo Fisher Catalog #16500100, GeneRuler 100 bp Plus DNA Ladder, ready-to-useThermo FisherScientificCatalog #SM0323, Ethidium bromide 10 mg / mlMerck MilliporeSigma (Sigma- Aldrich) Catalog #E1510, IX TAE Buffer Contributed by users.

[0106] A 5 pL of PCR product can be loaded on the agarose gel and electrophoresis is carried out at 120mV for about 30 minutes. A 3.5kb band should be seen in pool A and pool B reactions of each sample. Only samples for which both pool A and pool B reactions have worked can be taken forward for sequencing.

[0107] In an embodiment, in an optional step PCR products can be purified especially if primer dimers are detected in the PCR product. For this, AMPure XPBechman Coulter Catalog #A63882 bead slurry can be added to each sample in 1:1 volumetric ratio. It can be incubated at Room temperature for 00:15:00 to allow DNA binding to beads. The sample tubes can be kept on magnetic stand for separation of beads bound to DNA and supernatant can be carefully removed without disturbing the beads. It can be washed twice with 150 pL freshly prepared 80% Ethanol without disturbing the beads. It can be kept for drying in Room temperature for 00:00:30 to 00:01:00 to remove excess ethanol. The tube can be removed from magnetic stand and 25 pL of Nuclease Free Water (NFW) can be added. The beads can be mixed properly by pipetting and incubated for 15 minutes at room temperature to separate DNA from the beads. The tube can be kept on magnetic stand and wait for beads to separate out. The eluate (~20pl) containing DNA can be recovered into a fresh tube.

[0108] In an embodiment, the present invention provides a kit for amplification or detecting mutations in drug resistant genes of Plasmodium falciparum. The kit can comprise-

[0109] (i) a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof; and

[0110] (ii) a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof.

[0111] The kit can further comprise deoxynucleotide triphosphates (dNTPs), a buffer, DNA polymerase. The kit can optionally comprise a positive control containing synthetic DNA segments ofPT / 2026 / 11086

[0112] amplification targets. It can also optionally a negative control containing a reaction mixture without any nucleic acid. The kit can further comprise nuclease-free water.

[0113] The buffer may be 2x reaction buffer containing optimized concentrations of MgC12, dNTP’s, HiFi polymerase, hot start antibodies and ToughMix chemistry.

[0114] The DNA polymerase can be selected from but not limited to Quantabio repliQa HiFi ToughMix, Takara PrimeSTAR GXL, NEB Q5 Hot Start High-Fidelity 2X Master Mix, and NEB LongAmp Hot Start Taq DNA Polymerase. This corresponds to the genotyping of the P. falciparum genes implicated in drug resistance.

[0115] The kit may be supplied with instructions and reagents for multiplex PCR, enabling rapid detection.

[0116] In an embodiment the present invention also provides a kit for antimalarial screening of P. falciparum in a sample by MPCR and sequencing. The kit can comprise primer panel, as disclosed herein. The kit can optionally comprise a positive control containing synthetic DNA segments of amplification targets. It can also optionally a negative control containing a reaction mixture without any nucleic acid. The kit can further comprise nuclease-free water.

[0117] In an embodiment, the present invention relates to an assay for detecting mutations in drugresistance genes of Plasmodium falciparum. The method can comprise

[0118] a) contacting a sample suspected of containing Plasmodium falciparum DNA with a primer panel having pools of primers, each pool comprising:

[0119] a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof; and

[0120] a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof.

[0121] b) performing multiplex PCR to amplify target regions of drug-resistance genes having SEQ ID Nos:79-93;PT / 2026 / 11086

[0122] c) sequencing the amplified products; and

[0123] d) analyzing the sequence data to detect mutations in said drug-resistance genes.

[0124] In an embodiment, the present invention provides a method for the antimalarial surveillance of P. falciparum in a sample comprising:

[0125] a) contacting a sample with a primer panel having pools of primers, each pool comprising:

[0126] a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof; and

[0127] a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof.

[0128] b) performing multiplex PCR to amplify target regions of drug-resistance genes having SEQ ID Nos:79-93;

[0129] c) sequencing the amplified products.

[0130] In an embodiment, the present invention provides a method for antimalarial surveillance of P. falciparum in a sample by MPCR. The said method can comprise:

[0131] (i) obtaining a sample, said sample comprising target DNA sequence of P. falciparum-, (ii) denaturing the target DNA sequence from step (i); and

[0132] (iii) contacting said denatured target DNA with the primer panel as disclosed herein, deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to produce a first PCR product, followed by repeating the above steps till amplified copies of target nucleic acids are obtained.

[0133] In an embodiment of the present invention, the amplification of the target DNA in the reaction mixture under suitable PCR reaction mixture temperature conditions by a repetitive series of MPCR thermal cycling steps. The steps can comprise of:PT / 2026 / 11086

[0134] (i) denaturing the target DNA into opposite strands;

[0135] (ii) hybridizing the forward and reverse primers from the primer panel to the appropriate denatured strands; and

[0136] (iii) extending the hybridized primers with the four dNTPs and the nucleic acid polymerase.

[0137] In an embodiment of the present invention, the extension is effected by polymerase chain reaction (PCR). The extension or thermocycling parameters can also be varied to take advantage of the differential thermal stability between the forward primer and reverse primer. For example, following the denaturation step in thermocycling, an intermediate temperature may be introduced which is permissible for enriching primer binding but not for amplification primer binding; the temperature is then increased to the extension temperature (for example 72°C.), whereby permitting extension of the matched enriching primer and melting away of un-extended enriching primer. The cycles of an intermediate temperature and extension temperature can be repeated as many times as desirable to allow the matched primer to extend on as many target templates as possible. The temperature can then be reduced to permit amplification primer annealing and extension.

[0138] In an embodiment of the present invention, the present invention provides a method comprising a primary PCR cycle, wherein the primers are added in their respective tubes followed by a MPCR reaction consisting of denaturation, annealing, and extension / elongation amplification reactions.

[0139] PCR Cycle:

[0140] In an embodiment of the present invention, the initial denaturation and denaturation are effected at 95°C for 1 minute, followed by 35 cycles of 95°C denaturation for 15 seconds.

[0141] In an embodiment of the present invention, the annealing and extension is effected at 63 °C for 5 minutes.

[0142] In an embodiment of the present invention, the amplification reaction is selected from any of the polymerase chain reactions (PCRs) known in the prior art. Preferably, multiplex overlap PCR, which allows efficient amplification of all target genes in just two PCR reaction such as Pool A and Pool B as illustrated above per sample. This may enhance scalability and cost-effectiveness by reducing reaction complexity. The panel can generates 1.5 kb amplicons, and for genes exceeding this size, the amplicons are split into multiple fragments.PT / 2026 / 11086

[0143] In an embodiment of the present invention, the sample can a blood sample from humans. The sample can be a blood sample, EDTA Blood or Dried Blood Spot.

[0144] In an embodiment of the present invention, the robust detection of drug-resistant P. falciparum is achieved by the method disclosed herein even when the template for multiplexed PCR was spiked in at a low level of 0.05% parasetemia of total DNA.

[0145] In an embodiment of the present invention, the nucleotide sequence of the MPCR-amplified sequence can be determined using anyone or combination of the variety of methods for determining the nucleotide sequence of DNA. Preferably, Oxford Nanopore Technology (ONT).

[0146] In an embodiment of the present invention, the PfMDR15 designed for swift and reliable screening of antimalarial resistance from clinical samples of P. falciparum, proves valuable especially during seasonal outbreaks, and monitoring antimalarial efficacy in nominal cost. Moreover, it serves as a source for detection of the drug-resistant P. falciparum in a particular region which is very much needed for disease control and stopping the spread of malaria.

[0147] The method of the present invention can analyze up to 15 genes and detect both known and novel mutations. This method is also cost-effective and scalable. It uses a technique called multiplex overlap PCR, which may allow all target genes to be amplified in just two PCR reactions per sample. After that, a quick library preparation and sequencing process using Oxford Nanopore technology follows.

[0148] While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.

[0149] EXAMPLES

[0150] The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

[0151] EXAMPLE 1

[0152] Materials and Methods:PT / 2026 / 11086

[0153] Clinical P. falciparum malaria samples were received from Dr. Amit Sharma, International Center for Genetic Engineering and Biotechnology (ICGEB), New Delhi.The source of samples is blood from malaria positive patients. DNA extraction from the samples were done using the QIAGEN DNA Mini Kit. The extraction process followed a modified protocol as outlined on Malarigen.net.

[0154] Selective Whole Gene Amplification using Plasmodium falciparum Multi Drug Resistance 15 panel (P / MDR15)

[0155] PCR amplification of Drug resistance genes for P. falciparum from the samples is carried out for genotyping. The PCR amplification of 15 genes is done in multiplexed manner. The PCR primers were designed using the reference genome PlasmoDB68_Pfalciparum3D7 for Plasmodium falciparum available in PlasmoDB using PrimalScheme A total of 39 primer pairs were designed to cover all the 15 drug resistant genes and each PCR product is ~1.5kb in size PCR fragments. The Pf MDR15 primer set is divided into pool A and pool B such that adjacent amplicons are in different pools. The details of the entire primer set are given in below table:

[0156] Table 2: List and characteristics of Primers used in the present invention:

[0157]

[0158] PT / 2026 / 11086

[0159]

[0160] PT / 2026 / 11086

[0161]

[0162] PT / 2026 / 11086

[0163]

[0164] PT / 2026 / 11086

[0165]

[0166] For individual primers, 100 pmolar stocks were prepared in Nuclease Free Water and stored in - 20 °C. For multiplexed genomic PCR reaction, the pool A and pool B were prepared by separately mixing 10 pL of each of the pool A primers in one pool and 10 pL of each of the pool B primers in the other pool. The primer pools were stored in -20°C. At the time of setting up the PCRPT / 2026 / 11086

[0167] reactions the pool A and pool B primer pools were diluted 10 times (1 Opmolar primer mix) with Nuclease Free Water and added to the reactions as given in Table 3. Pool A and Pool B PCR reactions were set up separately using RepliQa HiFi ToughMix® VWR International Catalog #95200-500 master mix.

[0168] Table 3 - PCR reaction mix composition using PfMDR15

[0169]

[0170] PCR amplification conditions for genomic PCR reaction are given in Table 4

[0171] Table 4- PCR conditions for multiplex whole genome amplification using P / MDR15 panel

[0172]

[0173] Oxford Nanopore (ONT) Sequencing Steps Using SQK- RBK114.96 Kit

[0174] I. Barcoding

[0175] Between 50 ng to 100 ng of each purified PCR product was taken in maximum volume of 9 pL (make up volume with NFW if needed) and 1 pL sequencing rapid barcode (RB01-96) provided in the kit is added.

[0176] II. Purification of pooled barcoded sample

[0177] AM Pure XPBechman CoulterCatalog #A63882 bead slurry was added to each sample in 1:1 volumetric ratio. It was incubated at room temperature for 00:15:00 to allow DNA binding to beads. The sample tubes were kept on magnetic stand for separation of beads bound to DNA. The supernatant was carefully removed without disturbing the beads. It was washed twice with 1 mL freshly prepared 70% Ethanol without disturbing the beads. It was kept for drying in Room temperature for 30 seconds to remove excess ethanol. The tubes were removed from magneticPT / 2026 / 11086

[0178] stand and 20 pL of Elution Buffer (EB) provided in the kit was added. The beads were mixed properly by pipetting. It was incubated for 15 minutes at room temperature to separate DNA from the beads. The tube was kept on magnetic stand and waited for beads to separate out. The eluate (~15 pl) containing barcoded library was recovered into a fresh tube.

[0179] III. Adapter ligation of barcoded library

[0180] 11 pL of barcoded library was taken and 1 pL of diluted rapid adapter (1.5 pL Rapid Adapter (RA) + 3.5 pL Adapted Buffer (ADB)) was added. It was incubated at 37°C for 10 minutes and immediately proceeded with loading the library on the flowcell and sequencing.

[0181] IV. Flow cell priming

[0182] Pore scan of the flow cell followed by flowcell priming was performed as per ONT protocol. The priming mix was made by adding 30 pL Flow Cell Tether (FCT) in 1170 pL Flow Cell Flush (FCF) and mixed by vortexing. The waste was removed from waste port if required. 200 pL reading in 1000 pL pipette was set then OPEN priming port and air was sucked out by bringing pipette volume to 220 pL - 230 pL to avoid entry of air bubble while loading. 800 pL of priming mix was added via the priming port. It was incubated at room temperature for 05 minutes. Second priming of flow cell was done by adding 200 pL priming mix.

[0183] V. Sample preparation for loading

[0184] 37.5 pL Sequencing Buffer (SB) and 25.5 pL Library Beads (LIB) was added to adapter ligated sequencing library.

[0185] VI. Sequencing

[0186] Sequencing was done using MinKNOW software.

[0187] VII. Data Analysis

[0188] In-house bash script named as PFMDR15.sh was developed for automated data analysis, accessible from github via PFMDR.git link. All steps involved in data analysis and the corresponding programs are described in PFMDR.git.

[0189] Assay Standardization:

[0190] A total of 39 primer pairs were designed to target all 15 drug-resistance genes, with each PCR product measuring approximately 1.5 kb. Initially, P. falciparum (3D7) DNA from cultured cells was used to optimize the PCR conditions. The gel image (Figure 1) shows successful amplification of the individual genes under uniform PCR conditions, demonstrating the consistency of thePT / 2026 / 11086

[0191] process. Upon standardization and verification of the amplification of all individual amplicons, the products were pooled into two groups (Pools A and B), as described in the methodology.

[0192] The pooled amplicons were then subjected to PCR under the same standardized conditions, and the 1.5 kb bands were confirmed via gel electrophoresis. Following verification of amplification, sequencing was performed using Oxford Nanopore Technology (ONT), and the sequencing reads were mapped to a synthetic reference file created to represent all genes. Figure 2B displays the depth plot of the sequencing data, indicating successful amplification of all PCR amplicons and sufficient reads across all genes for comprehensive analysis. This 39-primer pair panel was named PfMDR15 (P. falciparum Multi-Drug-resistance 15).

[0193] To analyse specificity, the PfMDR15 panel was used to amplify DNA from both P. falciparum and human genomic templates. Figure 3 A shows a 1.5 kb band for P. falciparum DNA, while no band was observed with the human genomic DNA template. To confirm the integrity of the human DNA, control primers specific to human DNA were used, demonstrating that the human DNA was intact and confirming that the PfMDR15 panel is specific to P. falciparum and does not amplify human DNA. Sensitivity testing was performed by adjusting parasitaemia levels from 4% to 0.01% by mixing P. falciparum-infected red blood cells with whole blood from a healthy donor. Following dilution, the samples were spotted on DBS paper, DNA was extracted, and drugresistance markers were amplified using the PfMDR15 panel. Figure 3B shows the gel image with a 1.5 kb band following amplification, while Figure 4 presents the depth plots for parasitaemia levels of 4%, 0.05%, and 0.01%. The data indicate that at 0.05% parasitaemia, there was sufficient coverage across all genes for reliable analysis. Even at a parasitaemia level of 0.01%, adequate depth was observed across most genes, with minimal amplicon dropout. These results demonstrate the high sensitivity of the assay, capable of detecting as few as 5 parasites in 10,000 red blood cells, thereby providing sufficient data for analysis.

[0194] Advantages of the Invention

[0195] 1. The present invention provides an end-to-end method suitable for large scale surveillance to detect the prevalence of drug-resistant-P. falciparum, followed by sequencing using Oxford Nanopore Technology (ONT), and the protocol has been optimized as an easy-to- implement multiplexed drug-resistance detection and genotyping method, which is necessary for a robust and low-cost diagnosis and surveillance method.PT / 2026 / 11086

[0196] 2. Comprehensive Coverage: Unlike previous methods, this panel covers the full sequence of fifteen drug-resistance genes, not just partial regions.

[0197] 3. High Sensitivity: The method can detect mutations even when parasite levels are extremely low.

[0198] 4. Specificity: The primers are specific to P. falciparum and do not amplify human DNA.

[0199] 5. Scalability and Cost-Effectiveness: Only two PCR reactions are needed per sample, making the process efficient and suitable for large-scale surveillance.

[0200] 6. Kit Format: The described technology can also be provided as a kit, including all required reagents and controls for straightforward implementation in laboratories.

[0201] It can further enable health authorities and researchers to monitor the spread and evolution of drugresistant malaria parasites in an efficient and precise manner. The system may also support informed decisions regarding treatment policies and assists in tracking the effectiveness of antimalarial drugs across various regions, thereby contributing to improved malaria control and elimination initiatives.

Claims

PT / 2026 / 11086WE CLAIM:

1. A primer panel for amplification or detecting mutations in drug resistant genes of Plasmodium falciparum said primer panel having pools of primers, each pool comprising:(a) a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof; and(b) a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof.

2. The primer panel as claimed in claim 1, wherein the set of primers corresponds to one or more drug resistant genes having SEQ ID Nos:79-93.

3. A composition for amplification or detecting mutations in drug resistant genes of Plasmodium falciparum, wherein said composition comprises the primer panel as claimed in claim 1 and optionally comprising a master mix containing dNTPs, buffer, and a DNA polymerase.

4. A method for amplification of drug-resistant genes for Plasmodium falciparum in a sample by multiplex polymerase chain reaction (PCR), wherein the method comprises:(i) mixing half of the sample containing the target DNA with a primer pool containing a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof , a master mix comprising deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to obtain the PCR product;(ii) mixing the other half of the sample containing the target DNA with a primer pool containing a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof, a master mix comprising deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to obtain the PCR product; and(iii) processing the PCR products thus obtained for library preparation for sequencing.

5. The method as claimed in claim 6, the multiplex PCR comprises:PT / 2026 / 11086(i) denaturation at 90-99°C for 20-40 seconds, followed by one or more cycles of 90-99°C denaturation for 10-40 seconds, preferably, at 95°C; and(ii) annealing and extension at 40-70°C, preferably 63°C and holding at 4°C indefinitely.

6. The method as claimed in claim 6, wherein the amplicons in each of the PCR products is about 1.5 kb size having overlapping regions of about 0.5 kb size.

7. An assay for detecting mutations in drug-resistance genes of Plasmodium falciparum, comprising:a) mixing sample with a primer panel having pools of primers, each pool comprising:a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof; anda set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof.b) performing multiplex PCR to amplify target regions of drug-resistance genes having SEQ ID Nos:79-93;c) sequencing the amplified products; andd) analyzing the sequence data to detect mutations in said drug-resistance genes.

8. A method for the antimalarial surveillance of P. falciparum in a sample comprising:a) contacting a sample with a primer panel having pools of primers, each pool comprising:a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof; anda set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof.PT / 2026 / 11086b) performing multiplex PCR to amplify target regions of one or more drug resistant genes having SEQ ID Nos:79-93.c) sequencing the amplified products.

9. The method as claimed in claim 4-8, wherein the sample is a blood sample, EDTA blood or Dried Blood Spot (DBS).

10. A kit for amplification or detecting mutations in drug resistant genes of Plasmodium falciparum comprising:a. a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78 or variants thereof; andb. a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76 or variants thereof.c. deoxynucleotide triphosphates (dNTPs);d. a buffer;e. DNA polymerase;f. optionally, a positive control containing synthetic DNA segments of amplification targets;g. optionally a negative control containing a reaction mixture without any nucleic acid; andh. nuclease-free water.

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