Development of Two-Tube Loop-Mediated Isothermal Amplification Assay for Differential Diagnosis of Plasmodium falciparum and Plasmodium vivax and Its Comparison with Loopamp™ Malaria
Abstract
:1. Introduction
2. Materials and Methods
2.1. Study Area and Sample Collection
2.2. Nested PCR
2.3. LAMP Primer Design
2.4. Construction of Recombinant Plasmid
2.5. Closed-Tube LAMP Assay
2.6. Analytical Sensitivity and Specificity
2.7. Validation of LAMP Assay with Clinical Samples
2.8. LoopampTM Malaria Pan/Pf/Pv Detection Test
2.9. Statistical Analysis
3. Results
3.1. Validation of Clinical Sample by Nested PCR
3.2. Primers and Standardization of LAMP Assay
3.3. Analytical Sensitivity and Specificity of LAMP Assay
3.4. Sensitivity and Specificity of Two-Tube LAMP Assay in Clinical Samples
3.5. LoopampTM Malaria Detection Kit Performance
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Target Gene(s) | LAMP Assay | Detection Methods | Plasmodium Strain Used | Clinical Sample/Extraction Method | Sensitivity | Specificity | Time and Temperature for Positivity | Reference(s) |
---|---|---|---|---|---|---|---|---|
18S Ribosomal RNA gene sequence | OneStep turbidimetric conventional LAMP assay | Turbidimetric assay; naked eye | P. falciparum, P. vivax, P. malariae, P. ovale, P. knowlesi. yoelii | Heat-treated/extracted DNA from clinical blood samples | 95–98.5% | 94.3–99% | 60 °C for 30–120 min | [13,14] |
OneStep SYBR Green conventional LAMP assay | Fluorimetric assay-SYBR Green I and UV light; naked eye | Plasmodium spp., P. falciparum, P. vivax, P. malariae | Extracted parasite DNA from peripheral/placental blood, saliva and/or urine | 88.9–100% | 90–100% | 60–65 °C for 30–100 min | [15,16,17,18,19,20,21] | |
Malachite Green-LAMP WarmStart-LAMP | Colorimetric assay-malachite green dye and UV light; phenol red; naked eye | Plasmodium spp., P. falciparum, P. vivax, P. ovale, P. malariae | Parasite DNA extracted from clinical blood sample by the boil and spin method | 95–100% | 100% | 63 °C for 60 min | [22,23] | |
Real-Time fluorescence LAMP/ Multiplex microfluidic LAMP (mμLAMP) | Real-time fluorescence detector- SYTO-9/SYBR green amplification fluorescence peak/hydroxynaphthol blue (HNB) | P. vivax, P. falciparum | Clinical blood samples | 95–97% | 91–100% | 62–64 °C for 60–90 min | [24,25,26] | |
Mitochondrial DNA target (cox1 genes/cytochrome oxidase subunit 1 gene/others) | OneStep SYBR Green/Calcein conventional LAMP assay/LoopampTM Malaria Pan/Pf Detection kit (POC) | Fluorimetric assay-SYBR Green I/calcein and UV light; naked eye | Plasmodium spp., P. vivax, P. falciparum | Dried blood spot (DBS)/venous blood sample | 83.3–98% | 100% | 65 °C for 30–60 min | [27,28,29] |
Illumigene Malaria LAMP | Turbidometric assay | P. falciparum, P. vivax, P. ovale, P. malaria, P. knowlesi | Parasite DNA from whole blood | 95% | 95% | 62–65 °C for 60 min | [30] | |
High-Throughput, Loop-Mediated Isothermal Amplification (HtLAMP) | Colorimetric assay-hydroxynaphtholblue (HNB); naked eye | P. vivax | Parasite DNA from the whole blood sample. | 95% | 93% | 65 °C for 30 min | [31] | |
Real-Time fluorescence LAMP (OptiGene) | Fluorometrically assay-SYBR green; naked eye | P. vivax, P. falciparum, P. malariae, and P. ovale | Parasite DNA extracted from dried blood spots/dried saliva spots/urine | 90–96.7% | 85–91.7% | 63–65 °C for 30–90 min | [32] | |
Apical membrane antigen-1 (AMA-1) gene sequence | OneStep conventional LAMP | Fluorimetric assay- SYBR Green I; SYBR® Safe DNA gel stain and UV light; | P. knowlesi | Parasite DNA extracted from blood | 100% | 100% | 64 °C for 60 min | [33] |
Apicoplast genome | Conventional LAMP | naked eye | P. falciparum | Dried blood spot (DBS) sample | 92 | 97 | 65 °C for 60 min | [34] |
Exported protein 1 (PfExp1) | Reverse transcription fluorescence -LAMP | amplification fluorescence peak | P. falciparum | RNA from freed parasite pellets | 90% | Could not be determined * | 68 °C for 60 min | [35] |
Kit | Samples | LAMP Assay | ||||
---|---|---|---|---|---|---|
Cases Tested (Total) | Cases Positive (Total) | Sensitivity/Specificity (95% CI) | Positive Predictive Value | Negative Predictive Value | ||
Pf specific LAMP | Pf/Mix | 22/12 (34) | 22/12 (34) | 100% (89.72–100%) | 100% | 100% |
Pv/Other Disease/Healthy Control | 115/61/6 (182) | 0/0/0 (0) | 100% (97.99–100%) | |||
Pv specific LAMP | Pv/Mix | 115/12 (127) | 115/12 (127) | 100% (97.14–100%) | 100% | 100% |
Pf/Other Disease/Healthy Control | 22/61/6 (89) | 0/0/0 (0) | 100% (95.94–100%) |
Kit | Samples | Nested PCR Results | HUMAN LAMP Results | |||||
---|---|---|---|---|---|---|---|---|
Cases Tested (Total) | Cases Positive (Total) | Cases Tested (Total) | Cases Positive (Total) | Sensitivity/ Specificity (95% CI) | Positive Predictive Value (95% CI) | Negative Predictive Value (95% CI) | ||
Loopamp™ Malaria Pan Detection Kit | Pf/Pv/Mix | 15/15/8 (38) | 15/15/8 (38) | 15/15/8 (38) | 15/15/8 (38) | 100% (90.75–100%) | 100% | 100% |
Other Disease | 6 (6) | 0 (0) | 6 (6) | 0 (0) | 100% (54.07–100%) | |||
Loopamp™ Malaria Pf Detection Kit | Pf/Mix | 15/8 (23) | 15/8 (23) | 15/8 (23) | 15/8 (23) | 100% (85.18–100%) | 100% | 100% |
Pv/Other Disease | 15/6 (21) | 0/0 (0) | 15/6 (21) | 0/0 (0) | 100% (83.89–100%) | |||
Loopamp™ Malaria Pv Detection Kit | Pv/Mix | 53/8 (61) | 53/8 (61) | 53/8 (61) | 53/7 (60) | 98.39% (91.34–99.96%) | 100% | 96.43% (79.44–99.47%) |
Pf/Other Disease/Healthy Control | 15/6/6 (27) | 0/0/0 (0) | 15/6/6 (27) | 0/0/0 (0) | 100% (87.23–100%) |
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Azam, M.; Upmanyu, K.; Gupta, R.; Sruthy, K.S.; Matlani, M.; Savargaonkar, D.; Singh, R. Development of Two-Tube Loop-Mediated Isothermal Amplification Assay for Differential Diagnosis of Plasmodium falciparum and Plasmodium vivax and Its Comparison with Loopamp™ Malaria. Diagnostics 2021, 11, 1689. https://doi.org/10.3390/diagnostics11091689
Azam M, Upmanyu K, Gupta R, Sruthy KS, Matlani M, Savargaonkar D, Singh R. Development of Two-Tube Loop-Mediated Isothermal Amplification Assay for Differential Diagnosis of Plasmodium falciparum and Plasmodium vivax and Its Comparison with Loopamp™ Malaria. Diagnostics. 2021; 11(9):1689. https://doi.org/10.3390/diagnostics11091689
Chicago/Turabian StyleAzam, Mudsser, Kirti Upmanyu, Ratan Gupta, Karugatharayil Sasi Sruthy, Monika Matlani, Deepali Savargaonkar, and Ruchi Singh. 2021. "Development of Two-Tube Loop-Mediated Isothermal Amplification Assay for Differential Diagnosis of Plasmodium falciparum and Plasmodium vivax and Its Comparison with Loopamp™ Malaria" Diagnostics 11, no. 9: 1689. https://doi.org/10.3390/diagnostics11091689