The development of loop-mediated isothermal amplification targeting alpha-tubulin DNA for the rapid detection of Plasmodium vivax

Abstract

Background: Malaria that is caused by Plasmodium vivax is the most widely distributed human malaria. Its recent resurgence in many parts of the world, including the Republic of Korea (ROK), emphasizes the importance of improved access to the early and accurate detection of P. vivax to reduce disease burden. In this study, a rapid and efficient loop-mediated isothermal amplification (LAMP)-based method was developed and validated using blood samples from malaria-suspected patients.

Method: A LAMP assay targeting the α-tubulin gene for the detection of P. vivax was developed with six primers that recognize different regions of the target gene. The diagnostic performance of the α-tubulin LAMP assay was compared to three other tests: microscopic examinations, rapid diagnostic tests (RDTs), and nested polymerase chain reactions (PCRs) using 177 whole blood specimens obtained from ROK military personnel from May to December 2011.

Results: The α-tubulin LAMP assay was highly sensitive with a detection limit of 100 copies of P. vivax α-tubulin gene per reaction within 50 min. It specifically amplified the target gene only from P. vivax. Validation of the α-tubulin LAMP assay showed that the assay had the highest sensitivity (P < 0.001 versus microscopy; P = 0.0023 versus RDT) when nested PCR was used as the gold standard and better agreement (concordance: 94.9%, kappa value: 0.865) with nested PCR than RDT and microscopy. A Receiver Operation Characteristics analysis showed that the diagnostic accuracy of the α-tubulin LAMP assay for vivax malaria was higher (Area Under Curve = 0.908) than RDT and microscopy.

Conclusion: This study showed that the P. vivax α-tubulin LAMP assay, which can be used to diagnose early infections of vivax malaria, is an alternative molecular diagnostic tool and a point-of-care test that may help to prevent transmission in endemic areas.

Figure 1

Primer design for α-tubulin loop-mediated isothermal amplification (LAMP) assay for the detection of Plasmodium vivax. (A) The primers were selected based on a nucleotide sequence alignment of the target region of α-tubulin gene from P. vivax, P. falciparum [GenBank accession no. XM_001351490] and Plasmodium knowlesi [GenBank accession no. XM_002258237] using ClustalW software. Black boxes and bold fonts indicate identical and conserved nucleotides, respectively. The locations of the primer recognition sites are indicated by arrows with the primer names. The black arrowhead indicates the AatII cleavage site. F3, forward outer primer; B3, backward outer primer; FIP, forward inner primer; BIP, backward inner primer; LF, loop forward primer; and LB, loop backward primer. (B) Sequences of the α-tubulin LAMP primers.

Figure 2

Sensitivity and specificity of the P. vivax α-tubulin LAMP assay. (A) Sensitivity of the P. vivax α-tubulin LAMP assay. Serial 10-fold dilutions of the α-tubulin DNA (10, 10², 10³, and 10⁴ copies per reaction) were used for the LAMP assay, and the real-time amplification was monitored by a measurement of absorbance. (B) Correlation between the threshold time and the copy number of the α-tubulin DNA in serially diluted samples. The values on the y-axis are the threshold time (in min), which was defined as the time at which the threshold value of absorbance (0.1) was reached. The results show the means and standard deviations of three separate experiments. A plasmid containing no insert was used as a control. The LAMP products were visualized using (C) gel electrophoresis and (D) Loopamp® fluorescent detection reagent (FD). Lane M, a 100-bp molecular weight marker; lane 1, P. falciparum; lane 2, Plasmodium ovale curtisi; lane 3, Plasmodium ovale wallikeri; lane 4, Plasmodium malariae; lane 5, Plasmodium knowlesi; lane 6, P. vivax; lane 7, Babesia microti; lane 8, Toxoplasma gondii; lane 9, Cryptosporidium parvum; lane 10, Entamoeba histolytica; lane 11, Giardia lamblia; lane 12, Trichomonas vaginalis; lane 13; Acanthamoeba castellanii; lane 14, malaria-negative human DNA control; lane R, products of the AatII digestion of the LAMP product of α-tubulin.