Development of a Multiplex Loop-Mediated Isothermal Amplification Assay for Diagnosis of Plasmodium spp., Plasmodium falciparum and Plasmodium vivax
Abstract
:1. Introduction
2. Materials and Methods
2.1. Clinical Samples and DNA Extraction
2.2. Primer Design
2.3. Multiplex Malaria Pan/Pf/Pv/IC LAMP Assay
2.4. Real-Time Reverse Transcription (RT)-PCR
2.5. Limit of Detection (LOD) Tests of the Multiplex Malaria Pan/Pf/Pv/IC LAMP Assay
3. Results
3.1. Optimization of the Multiplex Malaria Pan/Pf/Pv/IC LAMP Primer Set
3.2. Limit of Detection (LOD) of the Multiplex Malaria Pan/Pf/Pv/IC LAMP Assay
3.3. Comparison of Clinical Performance between the Multiplex Malaria Pan/Pf/Pv/IC LAMP and RealStar® Malaria Screen & Type PCR Kit 1.0 Using Clinical Samples
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Target | Name | Sequence (5′-3′) | Length (mer) | Reference |
---|---|---|---|---|
Malaria pan (18S rRNA gene) | M. pan F3 | GTA TCA ATC GAG TTT CTG ACC | 21 | [31] |
M. pan B3 | CTT GTC ACT ACC TCT CTT CT | 20 | ||
M. pan FIP | TCG AAC TCT AAT TCC CCG TTA CCT ATC AGC TTT TGA TGT TAG GGT | 45 | ||
M. pan BIP | CGG AGA GGG AGC CTG AGA AAT AGA ATT GGG TAA TTT ACG CG | 41 | ||
M. pan FLP | CGT CAT AGC CAT GTT AGG CC | 20 | ||
M. pan BLP | AGC TAC CAC ATC TAA GGA AGG CAG | 24 | ||
M. pan BLP probe | [FAM]-CGG GCC CGT ACA AAG GGA ACA CCC ACA CTC CGA GCT ACC ACA TCT AAG GAA GGC AG | 56 | ||
Plasmodium falciparum (LDH gene) | P. f F3 | AAT TGT AAA CTT ACA TGC ATC AC | 23 | Present study |
P. f B3 | TCA AAT TTA GCT TTT TCC TCA CT | 23 | ||
P. f FIP | GCC CTT CTA ACA AGG TTG AGC AGC TGC TGC TAT TAT CGA AAT | 42 | ||
P. f BIP | TAT GGA CAC TCC GAT ATA TTC GGT GTA ATT CGA TAA CTT GTT CAA CAC | 48 | ||
P. f FLP | CAA ATC TTT AAG TAG GAT TCA GCC | 24 | ||
P. f BLP | GGT ACA CCT GTT GTT TTA GGT GCT | 24 | ||
P. f BLP probe | [HEX]-CGG GCC CGT ACA AAG GGA ACA CCC ACA CTC CGG GTA CAC CTG TTG TTT TAG GTG CT | 56 | ||
Plasmodium vivax (16S rRNA gene) | P. v F3 | GGA ATG ATG GGA ATT TAA AAC CT | 23 | [30] |
P. v B3 | ACG AAG TAT CAG TTA TGT GGA T | 22 | ||
P. v FIP | CTA TTG GAG CTG GAA TTA CCG CTC CCA AAA CTC AAT TGG AGG | 42 | ||
P. v BIP | AAT TGT TGC AGT TAA AAC GCT CGT AAG CTA GAA GCG TTG CT | 41 | ||
P. v FLP | GCT GCT GGC ACC AGA CTT | 18 | ||
P. v BLP | AGT TGA ATT TCA AAG AAT CG | 20 | ||
P. v BLP probe | [Texas red]-CGG GCC CGT ACA AAG GGA ACA CCC ACA CTC CGA GTT GAA TTT CAA AGA ATC G | 52 | ||
Human (actin gene) | IC F3 | AGT ACC CCA TCG AGC ACG | 18 | [32] |
IC B3 | AGC CTG GAT AGC AAC GTA CA | 20 | ||
IC FIP | GAG CCA CAC GCA GCT CAT TGT ATC ACC AAC TGG GAC GAC A | 40 | ||
IC BIP | CTG AAC CCC AAG GCC AAC CGG CTG GGG TGT TGA AGG TC | 38 | ||
IC FLP | TGT GGT GCC AGA TTT TCT CCA | 21 | ||
IC BLP | CGA GAA GAT GAC CCA GAT CAT GT | 23 | ||
IC BLP probe | [Cy5]-CGG GCC CGT ACA AAG GGA ACA CCC ACA CTC CGC GAG AAG ATG ACC CAG ATC ATG T | 55 | ||
Quencher probe 1 Quencher probe 2 | GAG TGT GGG TGT TCC CTT TGT ACG GGC CCG -BHQ1 | 30 | ||
CCT ACC CTC GTC CTA ACA CGG GAG CCT GCA CTG AC -BHQ2 | 35 |
Ct Values | |||||||||
---|---|---|---|---|---|---|---|---|---|
Vector Dilution (Copies/µL) | |||||||||
Primer set | Vector | 107 | 106 | 105 | 104 | 103 | 102 | 101 | |
Monoplex LAMP | Pan (FAM) | Malaria Pan | 8.08 | 9.10 | 10.21 | 11.04 | 12.82 | 16.26 | Neg |
Pf (HEX) | P. falciparum | 10.44 | 11.21 | 12.59 | 13.30 | 17.73 | 21.26 | Neg | |
Pv (Tex) | P. vivax | 12.05 | 14.22 | 15.48 | 16.00 | 19.77 | 22.60 | Neg | |
IC (Cy5) | IC | 14.26 | 16.18 | 18.00 | 19.62 | 24.28 | Neg | Neg | |
Multiplex LAMP | Pan (FAM) + Pf (HEX) + Pv (Tex) + IC (Cy5) | Malaria Pan | 12.15 | 13.78 | 15.55 | 17.28 | 19.10 | 22.81 | Neg |
P. falciparum | 17.79 | 19.00 | 20.53 | 26.65 | 31.48 | 31.67 | Neg | ||
P. vivax | 13.26 | 15.65 | 17.03 | 17.93 | 25.73 | 28.58 | Neg | ||
IC | 15.58 | 18.01 | 20.14 | 22.33 | 23.91 | Neg | Neg |
Clinical Samples | Whole Blood Clinical Sample | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Dilution * | 10−1 | 10−2 | 10−3 | 10−4 | 10−5 | 10−6 | ||||||||
CT | RFU | CT | RFU | CT | RFU | CT | RFU | CT | RFU | CT | RFU | |||
P. falciparum | Monoplex LAMP | Pan (FAM) | 9.86 | 15,145 | 10.83 | 16,032 | 12.68 | 17,075 | 14.43 | 17,203 | Neg | - | Neg | - |
Pf (HEX) | 9.71 | 16,795 | 10.82 | 16,581 | 13.73 | 17,218 | Neg | - | Neg | - | Neg | - | ||
Multiplex LAMP | Pan (FAM) | 16.70 | 7543 | 18.24 | 7629 | 20.70 | 8057 | 30.85 | 8118 | Neg | - | Neg | - | |
Pf (HEX) | 21.16 | 3928 | 22.88 | 3969 | 24.72 | 4108 | 38.28 | 4020 | Neg | - | Neg | - | ||
Pv (Tex) | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | ||
IC (Cy5) | 24.71 | 4603 | 37.16 | 2616 | Neg | - | Neg | - | Neg | - | Neg | - | ||
RealStar® Malaria S&T PCR Kit 1.0 | Pf (FAM) | 22.61 | 11,985 | 27.09 | 12,807 | 30.82 | 12,548 | 35.41 | 11,329 | 39.26 | 7558 | Neg | - | |
Pv (Cy5) | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | ||
P. vivax | Monoplex LAMP | Pan (FAM) | 16.12 | 13,226 | 18.05 | 14125 | 32.51 | 13,617 | 37.25 | 12,230 | Neg | - | Neg | - |
Pv (Tex) | 12.80 | 9561 | 14.59 | 9454 | Neg | - | Neg | - | Neg | - | Neg | - | ||
Multiplex LAMP | Pan (FAM) | 19.36 | 7347 | 20.90 | 7527 | 25.27 | 7546 | Neg | - | Neg | - | Neg | - | |
Pf (HEX) | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | ||
Pv (Tex) | 18.10 | 5398 | 20.08 | 5360 | 21.99 | 5541 | Neg | - | Neg | - | Neg | - | ||
IC (Cy5) | 54.86 | 1138 | 55.03 | 1124 | Neg | - | Neg | - | Neg | - | Neg | - | ||
RealStar® Malaria S&T PCR Kit 1.0 | Pf (FAM) | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | |
Pv (Cy5) | 29.37 | 5712 | 33.71 | 5585 | 36.90 | 5049 | Neg | - | Neg | - | Neg | - | ||
P. malariae | Monoplex LAMP | Pan (FAM) | 11.11 | 15,757 | 12.61 | 17,887 | 14.87 | 16,582 | 15.80 | 17,883 | Neg | - | Neg | - |
Multiplex LAMP | Pan (FAM) | 20.05 | 7706 | 22.29 | 7741 | 26.23 | 7531 | Neg | - | Neg | - | Neg | - | |
Pf (HEX) | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | ||
Pv (Tex) | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | ||
IC (Cy5) | 32.60 | 6815 | 37.41 | 6486 | 38.92 | 6449 | Neg | - | Neg | - | Neg | - | ||
RealStar® Malaria S&T PCR Kit 1.0 | Pm (FAM) | 24.56 | 13,326 | 28.16 | 12,145 | 31.22 | 1875 | 34.67 | 11,985 | 36.91 | 5500 | Neg | - | |
Po (Cy5) | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | ||
Pk (Tex) | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | ||
P. ovale | Monoplex LAMP | Pan (FAM) | 16.60 | 15,880 | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - |
Multiplex LAMP | Pan (FAM) | 22.20 | 7885 | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | |
Pf (HEX) | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | ||
Pv (Tex) | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | ||
IC (Cy5) | 26.27 | 6097 | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | ||
RealStar® Malaria S&T PCR Kit 1.0 | Pm (FAM) | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | |
Po (Cy5) | 34.12 | 2516 | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | ||
Pk (Tex) | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - | Neg | - |
Clinical Samples | Multiplex Malaria Pan/Pf/Pv/IC LAMP Assay | RealStar® Malaria Screen & Type PCR Kit 1.0 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Pan (FAM) | Pf (HEX) | Pv (Tex) | IC (Cy5) | Pf (FAM) | Pv (Cy5) | IC (HEX) | Pm (FAM) | Po (Cy5) | IC (HEX) | ||
P. falciparum (n = 61) | P/N | 61/0 | 61/0 | 0/61 | 46/15 | 61/0 | 1/60 | 58/3 | - | - | - |
Sensitivity | 100% | 100% | - | 75.41% | 100% | - | 95.08% | - | - | - | |
Specificity | - | - | 100% | - | - | 98.36% | - | - | - | - | |
P. vivax (n = 54) | P/N | 54/0 | 0/54 | 54/0 | 44/10 | 0/54 | 54/0 | 54/0 | - | - | - |
Sensitivity | 100% | - | 100% | 81.48% | - | 100% | 100% | - | - | - | |
Specificity | - | 100% | - | - | 100% | - | - | - | - | - | |
P. malariae (n = 2) | P/N | 2/0 | 0/2 | 0/2 | 2/0 | - | - | - | 2/0 | 0/2 | 2/0 |
Sensitivity | 100% | - | - | 100% | - | - | - | 100% | - | 100% | |
Specificity | - | 100% | 100% | - | - | - | - | - | 100% | - | |
P. ovale (n = 1) | P/N | 1/0 | 0/1 | 0/1 | 1/0 | - | - | - | 1/0 | 0/1 | 1/0 |
Sensitivity | 100% | - | - | 100% | - | - | - | 100% | - | 100% | |
Specificity | - | 100% | 100% | - | - | - | - | - | 100% | - | |
Non-infection (n = 90) | P/N | 0/90 | 0/90 | 0/90 | 90/0 | 0/90 | 0/90 | 90/0 | - | - | - |
Sensitivity | - | - | - | 100% | - | - | 100% | - | - | - | |
Specificity | 100% | 100% | 100% | - | 100% | 100% | - | - | - | - |
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Jang, W.S.; Lim, D.H.; Choe, Y.; Jee, H.; Moon, K.C.; Kim, C.; Choi, M.; Park, I.S.; Lim, C.S. Development of a Multiplex Loop-Mediated Isothermal Amplification Assay for Diagnosis of Plasmodium spp., Plasmodium falciparum and Plasmodium vivax. Diagnostics 2021, 11, 1950. https://doi.org/10.3390/diagnostics11111950
Jang WS, Lim DH, Choe Y, Jee H, Moon KC, Kim C, Choi M, Park IS, Lim CS. Development of a Multiplex Loop-Mediated Isothermal Amplification Assay for Diagnosis of Plasmodium spp., Plasmodium falciparum and Plasmodium vivax. Diagnostics. 2021; 11(11):1950. https://doi.org/10.3390/diagnostics11111950
Chicago/Turabian StyleJang, Woong Sik, Da Hye Lim, YoungLan Choe, Hyunseul Jee, Kyung Chul Moon, Chaewon Kim, Minkyeong Choi, In Su Park, and Chae Seung Lim. 2021. "Development of a Multiplex Loop-Mediated Isothermal Amplification Assay for Diagnosis of Plasmodium spp., Plasmodium falciparum and Plasmodium vivax" Diagnostics 11, no. 11: 1950. https://doi.org/10.3390/diagnostics11111950