WO2016120886A2 - A novel molecular diagnostic technique for detecting the different species of plasmodium - Google Patents
A novel molecular diagnostic technique for detecting the different species of plasmodium Download PDFInfo
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- WO2016120886A2 WO2016120886A2 PCT/IN2016/000023 IN2016000023W WO2016120886A2 WO 2016120886 A2 WO2016120886 A2 WO 2016120886A2 IN 2016000023 W IN2016000023 W IN 2016000023W WO 2016120886 A2 WO2016120886 A2 WO 2016120886A2
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- plasmodium
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- pcr
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- 241000224016 Plasmodium Species 0.000 title claims abstract description 20
- 241000894007 species Species 0.000 title claims abstract description 13
- 238000012631 diagnostic technique Methods 0.000 title description 2
- 238000000034 method Methods 0.000 claims abstract description 37
- 230000008569 process Effects 0.000 claims abstract description 14
- 241000223960 Plasmodium falciparum Species 0.000 claims abstract description 8
- 241001505293 Plasmodium ovale Species 0.000 claims abstract description 7
- 206010035502 Plasmodium ovale infection Diseases 0.000 claims abstract description 6
- 241000223810 Plasmodium vivax Species 0.000 claims abstract description 5
- 230000029087 digestion Effects 0.000 claims description 14
- 108091008146 restriction endonucleases Proteins 0.000 claims description 12
- 230000003321 amplification Effects 0.000 claims description 8
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 8
- 238000012163 sequencing technique Methods 0.000 claims description 8
- 108020004414 DNA Proteins 0.000 claims description 5
- 108020005196 Mitochondrial DNA Proteins 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 18
- 230000002438 mitochondrial effect Effects 0.000 description 11
- 239000012634 fragment Substances 0.000 description 9
- 238000007857 nested PCR Methods 0.000 description 9
- 201000004792 malaria Diseases 0.000 description 8
- 238000003745 diagnosis Methods 0.000 description 6
- 244000045947 parasite Species 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000012124 rapid diagnostic test Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 230000004544 DNA amplification Effects 0.000 description 3
- 238000001712 DNA sequencing Methods 0.000 description 3
- 238000007397 LAMP assay Methods 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000386 microscopy Methods 0.000 description 3
- 238000007399 DNA isolation Methods 0.000 description 2
- 241000223821 Plasmodium malariae Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 2
- 108020004463 18S ribosomal RNA Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- 206010035501 Plasmodium malariae infection Diseases 0.000 description 1
- 206010035503 Plasmodium vivax infection Diseases 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229940118768 plasmodium malariae Drugs 0.000 description 1
- 244000000040 protozoan parasite Species 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 108020004418 ribosomal RNA Proteins 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- This invention relates to a process for detecting different species of Plasmodium. BACKGROUND OF THE INVENTION
- PCR polymerase chain reaction
- It is a further objection of this invention is to propose a process for detecting different species of Plasmodium, which is simple and cost-effective.
- Yet another objection of this invention is to propose a process for detecting different species of Plasmodium, which is sensitive and superior to known PCR techniques.
- FIGURE 1 Gel picture showing mitochondrial genome amplified product. Lane 1-100 bp marker and Lane 2-9 showing amplified fragment of mitochondrial gene.
- FIGURE 2 Mitochondrial fragment after digestion with restriction enzymes Mlucl and Taql
- reference samples and field and clinic samples are collected, and the genomic, DNA is isolated followed by Plasmodium mitochondrial gene amplification by PCR-RFLP.
- the amplied PCR product is then subjected to digestion with restriction enzyme and sequenced. Sequencing is used for validation purpose to check if the correct band is amplified or not, but the procedure does not require sequencing for confirmation.
- Reference Sample Reference samples of Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae were taken from parasite bank facility of NIMR, New Delhi. Plasmodium ovale sample were from Filter spots (QC samples) sent under WHO accreditation of QC laboratory.
- Field and Clinic Sample Fifty samples were collected from Malaria Clinic of NIMR, New Delhi (between June-Aug. 2014) and fifty samples were taken from study site at Betul district of Madhya Pradesh where Therapeutic efficacy studies were conducted during 2011-2012.
- Genomic DNA was isolated from blood spots using QIAamp DNA minikit, (Qiagen, Germany) according to manufacturing protocol with slight modifications.
- PCR amplification Plasmodium mitochondrial gene amplification was performed in 20 ⁇ reaction mixture containing 8.5 ⁇ nuclease-free water, 2 ⁇ of genomic DNA, 2 ⁇ ⁇ PCR buffer (Life Technologies, Inc.), 3.2 ⁇ of 25 mM MgC (Life Technologies, Inc.), 2 ⁇ of 10 mM deoxynucleotide triphosphates (dNTP, Bangalore Genei), 1 ⁇ of each forward and reverse primer (lOpmol) (GCC, biotech India) and 0.3 ⁇ 1 of 5 U/ ⁇ of AmpliTaq Gold® DNA Polymerase (Life Technologies, Inc.).
- the mitochondrial genome of Plasmodium was amplified using the forward primer 5 '-TCGCTTCTAACGGTGAAC -3 ' and reverse primer 5'- AATTGATAGTATCAGCTATCCATAG-3 '. Reactions were carried out in a Thermal cycler (Applied Biosystem).
- Applied Biosystem For Plasmodium mitochondrial gene amplification conditions were as follows: Step 1 , 95°C for 10 min; step 2, 95°C for 30 Second; step 3, 62°C for 20 second; step 4, 72°C for 15 second, repeat steps 2-4, 50 times, and final extension at 72°C for 10 min.
- Digested PCR product was analyzed on 2.5% agarose gel containing ethidium bromide (0.5 g/ml) and 0.5X TBE running buffer (pH 8.0). Digested PCR products were captured with the help of gel documentation system.
- 220bp Mitochondrial region of Plasmodium species was amplified at 220bp fragment (Fig.l). Digestion with Mlucl restriction enzyme leads to fragment sizes for different species; 216 bp fragment for vivax, 139bp + 49bp + 27bp fragment for malariae and 189bp + 27bp fragment for falciparum/ovale. To distinguish P. falciparum and P.ovale amplified 220bp product was digested with Taql restriction enzyme which leads to digestion of ovale of fragment size 147bp and 78bp while falciparum was undigested with fragment size 220bp (Fig.2 & Table 2). The sequencing results further confirmed the results.
- the invention provides a simple, less time consuming and very cost effective method for distinction of four human Plasmodium species namely P. falciparum, P. ovale, P.vivax, P. malariae, while other methods such as nested PCR, real time PCR and sequencing requires more costly chemicals and they are comparatively more time consuming and tedious.
- a comparison between nested PCR and the novel PCR technique according to the invention shows the improvements (Table 4).
- Target gene 18s rRNA Mitochondrial (MT) gene (other techniques like LAMP (Complicated primer design) & other method also target this gene (Needs sequencing to differentiate between four species). Assays using mitochondrial targets show higher sensitivity given the higher copy number.
- MT Mitochondrial
- Nested PCR Quality of Nested PCR is usually Nested PCR is not required (only single result carried out that step PCR is done).
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Abstract
This invention relates to a process for detecting different species of Plasmodium such as P. falciparum, P. vivax, P. malarie and P. ovale.
Description
A NOVEL MOLECULAR DIAGNOSTIC TECHNIQUE FOR DETECTING THE DIFFERENT
SPECIES OF PLASMODIUM
FIELD OF THE INVENTION
This invention relates to a process for detecting different species of Plasmodium. BACKGROUND OF THE INVENTION
More than 0.88 million people are infected by malaria parasites in India [1]. Rapid and accurate diagnosis of malaria is essential to decrease morbidity and mortality caused by protozoan parasite Plasmodium. The routinely used methods for diagnosis of malaria include microscopy and Rapid diagnostic tests (RDTs). But these two methods are faced with a number of challenges like requirement of experienced manpower, good quality of equipment and ambient storage conditions etc. Various molecular methods have been developed and used, such as polymerase chain reaction (PCR), which aims at increasing the sensitivity and specificity of diagnosis of malaria parasite [2] however, they too have certain limitations. Since the introduction of PCR for malaria diagnosis, many processes have been evolved with new amplification methods such as Real-Time PCR and loop-mediated isothermal amplification (LAMP) [3, 4]. Although both of these methods have benefit over conventional PCR in turnaround time, the nested PCR originally described by Snounou et al. in 1993 [5] and improved in 1999 [6], continues to be the gold standard /reference method. However, limitations of nested PCR include its time-consuming operation, substantial risk of contamination and high cost-efficiency. The nested PCR targets small subunit of ribosomal RNA (18s locus) with a sensitivity of 1 to 10 parasites per microlitre (1-10 ρ/μΐ) [5], whereas a sensitivity of 5 ρ/μΐ was achieved using an amplification target on the 6kb mitochondrial genome [7]. Recently one study has used mitochondrial genome to identify different species, however it employed DNA sequencing to confirm the diagnosis [8]. DNA Sequencing itself is tedious, costly and time consuming process.
Therefore, the need exists in the art to develop a Plasmodium species specific PCR assays, which is based on previously described primers targeting the mitochondrial genome.
OBJECTS OF THE INVENTION
It is, therefore, an object of this invention to propose a process for detecting different species of Plasmodium.
It is a further objection of this invention is to propose a process for detecting different species of Plasmodium, which is simple and cost-effective.
Yet another objection of this invention is to propose a process for detecting different species of Plasmodium, which is sensitive and superior to known PCR techniques.
These and other objections and advantages of the invention will be apparent from the ensuing description.
BRIEF DESCRIPTION OF THE ACCOMPANYING DIAGRAMS
FIGURE 1: Gel picture showing mitochondrial genome amplified product. Lane 1-100 bp marker and Lane 2-9 showing amplified fragment of mitochondrial gene.
FIGURE 2: Mitochondrial fragment after digestion with restriction enzymes Mlucl and Taql
DETAILED DESCRIPTION OF THE INVENTION:
Thus according to this invention is provided a process for detecting different species of Plasmodium.
In accordance with this invention, reference samples and field and clinic samples are collected, and the genomic, DNA is isolated followed by Plasmodium mitochondrial gene amplification by PCR-RFLP. The amplied PCR product is then subjected to digestion with restriction enzyme and sequenced. Sequencing is used for validation purpose to check if the correct band is amplified or not, but the procedure does not require sequencing for confirmation.
The invention will now be explained in greater details with the help of the following non-limiting example.
EXAMPLES
Reference Sample: Reference samples of Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae were taken from parasite bank facility of NIMR, New Delhi. Plasmodium ovale sample were from Filter spots (QC samples) sent under WHO accreditation of QC laboratory.
Field and Clinic Sample: Fifty samples were collected from Malaria Clinic of NIMR, New Delhi (between June-Aug. 2014) and fifty samples were taken from study site at Betul district of Madhya Pradesh where Therapeutic efficacy studies were conducted during 2011-2012.
Genomic Isolation: Genomic DNA was isolated from blood spots using QIAamp DNA minikit, (Qiagen, Germany) according to manufacturing protocol with slight modifications.
PCR amplification: Plasmodium mitochondrial gene amplification was performed in 20 μΐ reaction mixture containing 8.5 μΐ nuclease-free water, 2 μΐ of genomic DNA, 2 μΐ ΙΟχ PCR buffer (Life Technologies, Inc.), 3.2 μΐ of 25 mM MgC (Life Technologies, Inc.), 2 μΐ of 10 mM deoxynucleotide triphosphates (dNTP, Bangalore Genei), 1 μΐ of each forward and reverse primer (lOpmol) (GCC, biotech India) and 0.3μ1 of 5 U/ μΐ of AmpliTaq Gold® DNA Polymerase (Life Technologies, Inc.). The mitochondrial genome of Plasmodium was amplified using the forward primer 5 '-TCGCTTCTAACGGTGAAC -3 ' and reverse primer 5'- AATTGATAGTATCAGCTATCCATAG-3 '. Reactions were carried out in a Thermal cycler (Applied Biosystem). For Plasmodium mitochondrial gene amplification conditions were as follows: Step 1 , 95°C for 10 min; step 2, 95°C for 30 Second; step 3, 62°C for 20 second; step 4, 72°C for 15 second, repeat steps 2-4, 50 times, and final extension at 72°C for 10 min. (Reaction time: only 1 hour and 30 min)
Digestion with restriction enzymes: Amplified PCR product was digested with Mlucl (NEB, Inc.) restriction enzymes for the identification of Plasmodium vivax, P. malaria and P. faldparum/P. o ale. Taql (NEB, Inc.) restriction enzyme was used to distinguish P. falciparum and P. ovale (Reaction time: 20-25 min).
Digested PCR product was analyzed on 2.5% agarose gel containing ethidium bromide (0.5 g/ml) and 0.5X TBE running buffer (pH 8.0). Digested PCR products were captured with the help of gel documentation system.
Sequencing: In order to standardize the method the inventors have validated the result by sequencing. For this, the amplified 220bp PCR products of species specific positive samples were sequenced in both directions using forward and reverse primers. The PCR products were purified with Qiagen purification Kit according to the manufacturer's instructions. Purified PCR products were sent to Macrogen Inc, Korea for DNA sequencing. DNA sequences were edited and aligned (CmstaTW method) with Blast.
Ethics: All sample used in the study were taken from studies of NIMR which had ethical approval vide ethics meeting held on 24.07.2014.
The details of methodology and the primers used are shown in Table- 1.
Table !
Primers Amplification Digestion With MluCI Digestion
With Taql
Forward - 95°C for 5min, Pv Pm Pf/Po Pf Po
5'-TCGCTTCTAACGGTGAAC-3 ' 62 for 20 sec,
Reverse72 for 15 sec, 216+4 139+49+ 189+2+ 220 147 s' AATTGATAGTATCAGCTATC Total 50 cycles 27+4 7+4 +78
CATAG-3' Product size:
220bp
Mitochondrial region of Plasmodium species was amplified at 220bp fragment (Fig.l). Digestion with Mlucl restriction enzyme leads to fragment sizes for different species; 216 bp fragment for vivax, 139bp + 49bp + 27bp fragment for malariae and 189bp + 27bp fragment for falciparum/ovale. To distinguish P. falciparum and P.ovale amplified 220bp product was digested with Taql restriction enzyme which leads to digestion of ovale of fragment size 147bp and 78bp while falciparum was undigested with fragment size 220bp (Fig.2 & Table 2). The sequencing results further confirmed the results.
Summary of analysis of samples with microscopy, RDT and molecular diagnosis (Mitochondria gene) are shown in Table- 2.
Table-2
This technique was applied onto the field samples collected from site at Betul district of Madhya Pradesh and Malaria Clinic of NIMR, New Delhi (between June-Aug. 2014). All these samples were diagnosed by three different methods and comparative results of each were tabulated in Table 3. It was observed that the MtPCR technique according to the invention was much more sensitive and specific as compared to microscopy and RDT technique.
Comparison between nested PCR and Novel PCR
Table-4
S. No. Features Nested PCR method Novel PCR-RFLP method
1 Target gene 18s rRNA Mitochondrial (MT) gene (other techniques like LAMP (Complicated primer design) & other method also target this gene (Needs sequencing to differentiate between four species). Assays using mitochondrial targets show higher sensitivity given the higher copy number.
2 Principle Amplification of Amplification of mitrochondrial gene (MT conserved sequence gene) and further digestion with specific for different restriction enzymes
Plasmodium Species
3 Procedure 6 PCR reaction 1 PCR reaction + restriction digestion required required with only Two restriction enzymes (2 RE)
4 Total duration 25-30 hours 3-4 hours
of the technique
to get the result
5 Detection limit 10 parasite/ μΐ of blood 0.5 parasite/μΐ of blood
6 Quality of Nested PCR is usually Nested PCR is not required (only single result carried out that step PCR is done).
increase the chances of
contamination.
7 Cost per sample 540 rupees (cost for 120 rupees (cost for DNA isolation is
DNA isolation is excluded)
excluded)
Claims
1. A process for detecting different species of Plasmodium such as P. falciparum, P. vivax, P. malane and P. ovale.
2. The process as claimed in claim 1 , comprising the steps of isolating the genomic DNA of Plasmodium species, subjecting the Plasmodium mitochondrial gene to amplification using a forward primer and a reverse primer to lead to an amplified product, followed by digestion of the amplified product with a restriction enzyme for the identification of the different Plasmodium species and analysis of the product, followed by sequencing the amplified PCR products.
3. The process as claimed in claim 2, wherein said step of amplification comprises the following steps:
Step 1 : 95°C for 10 minutes
Step 2: 95°C for 30 seconds
Step 3: 62°C for 20 seconds
Step 4: 72°C for 15 seconds
followed by repeating steps 2-4 50 times and final extension at 72°C for 10 minutes.
4. The process as claimed in claim 1, wherein primers used are:
Primers Amplification Digestion With Ml CI Digestion
With Taql
Forward- 95°C for 5min, Pv Pm Pf/Po Pf Po
5'-TCGCTTCTAACGGTGAAC-3' 62 for 20 sec,
Reverse72 for 15 sec, 216+4 139+49+ 189+2+ 220 147 s' AATTGATAGTATCAGCTATC Total 50 27+4 7+4 +78
CATAG-3' cycles Product
size: 220bp
5. The process as claimed in claim 1 , wherein for the step of digestion, MlucI restriction enzyme is used for P. νίυαχ, P. malana and P. falciparum.
6. The process as claimed in claim 1, wherein for the step of digestion, TaqI restriction enzyme is used for P. falciparum and P. ovale.
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| Application Number | Priority Date | Filing Date | Title |
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| IN268/DEL/2015 | 2015-01-30 | ||
| IN268DE2015 | 2015-01-30 |
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Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998035057A1 (en) * | 1997-02-06 | 1998-08-13 | The National University Of Singapore | Diagnosis of plasmodium infection by analysis of extrachromosomal genetic material |
| US7927801B2 (en) * | 2006-11-30 | 2011-04-19 | Id-Fish Technology, Inc. | Nucleic acid probes and methods for detecting Plasmodium parasites |
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2016
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