Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis
Isothermal techniques with lateral flow detection have emerged as a point of care (POC) technique for malaria, a major parasitic disease in tropical countries such as India. <i>Plasmodium falciparum</i> and <i>Plasmodium vivax</i> are the two most prevalent malaria species fo...
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MDPI AG
2021-12-01
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| Series: | Diagnostics |
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| Online Access: | https://www.mdpi.com/2075-4418/12/1/32 |
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| author | Supriya Sharma Sandeep Kumar Md Zohaib Ahmed Nitin Bhardwaj Jaskirat Singh Sarita Kumari Deepali Savargaonkar Anupkumar R. Anvikar Jyoti Das |
| author_facet | Supriya Sharma Sandeep Kumar Md Zohaib Ahmed Nitin Bhardwaj Jaskirat Singh Sarita Kumari Deepali Savargaonkar Anupkumar R. Anvikar Jyoti Das |
| author_sort | Supriya Sharma |
| collection | DOAJ |
| description | Isothermal techniques with lateral flow detection have emerged as a point of care (POC) technique for malaria, a major parasitic disease in tropical countries such as India. <i>Plasmodium falciparum</i> and <i>Plasmodium vivax</i> are the two most prevalent malaria species found in the country. An advanced multiplex loop-mediated isothermal amplification (mLAMP) combined with a lateral flow dipstick (LFD) technique was developed for the swift and accurate detection of <i>P. falciparum</i> and <i>P. vivax</i>, overcoming the challenges of the existing RDTs (rapid diagnostic tests). A single set of LAMP primers with a biotinylated backward inner primer (BIP primer) was used for DNA amplification of both malaria species in a single tube. The amplified DNA was hybridized with fluorescein isothiocyanate (FITC) and digoxigenin-labelled DNA probes, having a complemented sequence for the <i>P. falciparum</i> and <i>P. vivax</i> genomes, respectively. A colour band appeared on two separate LFDs for <i>P. falciparum</i> and <i>P. vivax</i> upon running the hybridized solution over them. In total, 39 clinical samples were collected from ICMR-NIMR, New Delhi. Melting curve analysis, with cross primers for both species, was used to ascertain specificity, and the sensitivity was equated with a polymerase chain reaction (PCR). The results were visualized on the LFD for both species within 60 min. We found 100% sensitivity and specificity, when compared with a traditional PCR. Melting curve analysis of mLAMP revealed the lowest detection limit of 0.15 pg/μL from sample genomic DNA. The mLAMP-LFD assays could be a potential point of care (POC) tool for early diagnosis in non-laboratory conditions, with the convenience of a reduced assay time and the simple interpretation of results. |
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| institution | Directory Open Access Journal |
| issn | 2075-4418 |
| language | English |
| last_indexed | 2024-03-10T01:39:13Z |
| publishDate | 2021-12-01 |
| publisher | MDPI AG |
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| series | Diagnostics |
| spelling | doaj.art-3f0b6a8a3716470293bd105025cb0bfb2023-11-23T13:27:06ZengMDPI AGDiagnostics2075-44182021-12-011213210.3390/diagnostics12010032Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve AnalysisSupriya Sharma0Sandeep Kumar1Md Zohaib Ahmed2Nitin Bhardwaj3Jaskirat Singh4Sarita Kumari5Deepali Savargaonkar6Anupkumar R. Anvikar7Jyoti Das8Parasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, IndiaParasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, IndiaParasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, IndiaParasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, IndiaParasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, IndiaParasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, IndiaParasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, IndiaParasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, IndiaParasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, IndiaIsothermal techniques with lateral flow detection have emerged as a point of care (POC) technique for malaria, a major parasitic disease in tropical countries such as India. <i>Plasmodium falciparum</i> and <i>Plasmodium vivax</i> are the two most prevalent malaria species found in the country. An advanced multiplex loop-mediated isothermal amplification (mLAMP) combined with a lateral flow dipstick (LFD) technique was developed for the swift and accurate detection of <i>P. falciparum</i> and <i>P. vivax</i>, overcoming the challenges of the existing RDTs (rapid diagnostic tests). A single set of LAMP primers with a biotinylated backward inner primer (BIP primer) was used for DNA amplification of both malaria species in a single tube. The amplified DNA was hybridized with fluorescein isothiocyanate (FITC) and digoxigenin-labelled DNA probes, having a complemented sequence for the <i>P. falciparum</i> and <i>P. vivax</i> genomes, respectively. A colour band appeared on two separate LFDs for <i>P. falciparum</i> and <i>P. vivax</i> upon running the hybridized solution over them. In total, 39 clinical samples were collected from ICMR-NIMR, New Delhi. Melting curve analysis, with cross primers for both species, was used to ascertain specificity, and the sensitivity was equated with a polymerase chain reaction (PCR). The results were visualized on the LFD for both species within 60 min. We found 100% sensitivity and specificity, when compared with a traditional PCR. Melting curve analysis of mLAMP revealed the lowest detection limit of 0.15 pg/μL from sample genomic DNA. The mLAMP-LFD assays could be a potential point of care (POC) tool for early diagnosis in non-laboratory conditions, with the convenience of a reduced assay time and the simple interpretation of results.https://www.mdpi.com/2075-4418/12/1/32malariadiagnosismultiplex loop-mediated isothermal amplification (mLAMP)lateral flow detection (LFD)point of care (POC) |
| spellingShingle | Supriya Sharma Sandeep Kumar Md Zohaib Ahmed Nitin Bhardwaj Jaskirat Singh Sarita Kumari Deepali Savargaonkar Anupkumar R. Anvikar Jyoti Das Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis Diagnostics malaria diagnosis multiplex loop-mediated isothermal amplification (mLAMP) lateral flow detection (LFD) point of care (POC) |
| title | Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis |
| title_full | Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis |
| title_fullStr | Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis |
| title_full_unstemmed | Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis |
| title_short | Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis |
| title_sort | advanced multiplex loop mediated isothermal amplification mlamp combined with lateral flow detection lfd for rapid detection of two prevalent malaria species in india and melting curve analysis |
| topic | malaria diagnosis multiplex loop-mediated isothermal amplification (mLAMP) lateral flow detection (LFD) point of care (POC) |
| url | https://www.mdpi.com/2075-4418/12/1/32 |
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