Loop-Mediated Isothermal PCR (LAMP) for the Diagnosis of Falciparum Malaria

Paris, Daniel; Imwong, Mallika; Faiz, Abul; Hasan, Mahtabuddin; Yunus, Emran Bin; Silamut, Kamolrat; Lee, Sue J.; Day, Nicholas P. J.; Dondorp, Arjen M.

doi:10.4269/ajtmh.2007.77.972

Cited by 123 publications

(105 citation statements)

References 20 publications

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Section: Discussionmentioning

confidence: 99%

“…This higher sensitivity of LAMP compared to PCR has been described elsewhere (Han & Ge 2008). Moreover, the greater sensitivity of LAMP with crude lysate might indicate that LAMP is more robust with respect to dirty samples, because the LAMP method uses a robust DNA polymerase with low sensitivity to inhibitors (Paris et al 2007).Sensitive and specific assays that can be performed efficiently on infected fish tissue in a clinical laboratory are essential for early detection of pathogens and appropriate and effective antibiotic therapy. Bacteria were isolated from spleen, kidney and brain of all experimentally infected tilapia and grey mullet and identified as Lactococcus garvieae by PCR using pLG-1 and pLG-2.…”

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Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Tsai¹,

Wang²,

Yoshida³

et al. 2013

Dis. Aquat. Org.

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Based on use of a loop-mediated isothermal amplification (LAMP) identification protocol, this study attempted to detect Lactococcus garvieae in fish by using primer sets designed from an L. garvieae alpha/beta fold family hydrolase gene. Reaction time and temperatures were optimized for 60 min at 60°C with the resulting amplicons visualized by adding SYBR Green I to the reaction tube. The assay specificity was assessed using 45 different bacterial strains. Positive results were observed in all 30 L. garvieae isolates from various aquatic animals. No false-positive results were observed in 15 non-L. garvieae strains. The detection limit of the LAMP assay was 10-fold more sensitive than the conventional polymerase chain reaction (PCR) targeting 16S rDNA when using purified L. garvieae DNA. The detection limit of the LAMP assay was approximately 300 colony-forming units (CFU) using crude bacterial lysates, 100-fold more sensitive than PCR. Furthermore, L. garvieae in spleen, kidney and brain of experimentally challenged tilapia and grey mullet were detected using this optimized LAMP assay. Results of this study demonstrate the effectiveness of LAMP in providing a rapid yet simple test for detecting L. garvieae in fish.KEY WORDS: Lactococcosis · Alpha/beta fold family · Hydrolase gene · Loop-mediated isothermal amplification · Polymerase chain reaction · SYBR Green I Resale or republication not permitted without written consent of the publisherDis Aquat Org 102: [225][226][227][228][229][230][231][232][233][234][235] 2013 swimming. However, similar clinical signs are also found in Streptococcus sp. infections (Eldar & Ghittino 1999).Molecular detection methods of Lactococcus garvieae have been developed, including polymerase chain reaction (PCR) (Zlotkin et al. 1998, Aoki et al. 2000, Pu et al. 2002 and 16S rRNA and sodA sequence analysis (Fihman et al. 2006). However, although considered rapid and specific methods for detection, these require expensive equipment and other costly materials (Gunimaladevi et al. 2005).A method of amplifying nucleic acid sequences with high specificity, sensitivity and rapidity under isothermal conditions (60 to 65°C), loop-mediated isothermal amplification (LAMP) has been developed more recently (Notomi et al. 2000) and applied to fish pathogen detection (Caipang et al. 2004, Savan et al. 2004, Yeh et al. 2005, Itano et al. 2006. The assay uses a set of 4 or 6 primers that create a targetspecific stem loop DNA structure during initial amplification steps; subsequent LAMP auto-cycling is performed by the Bst DNA polymerase large fragment (Notomi et al. 2000). Since it does not require complicated equipment, the LAMP method provides a cost-effective, simple, and rapid (producing a result within 60 min) DNA amplification method.Recently, a fosmid library of Lactococcus garvieae from grey mullet has been constructed and many new genes of L. garvieae were cloned (M. A. Tsai et al. unpubl. data). The clone flg08-6 contained an alpha/beta fold family hydrolase gene that is also...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Tsai¹,

Wang²,

Yoshida³

et al. 2013

Dis. Aquat. Org.

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“…The specificity of microscopy was lower than previously reported. 22 Only one sample was non-concordant for P. vivax between LAMP and nPCR. This sample was diagnosed with a mixed infection of P. vivax and P. falciparum by nPCR and a single P. falciparum infection by LAMP.…”

Section: Discussionmentioning

confidence: 99%

“…As shown previously, even simple heat-treated blood can be used for the assay without further purification as it has been reported for falciparum malaria. 22 Furthermore, in larger hospitals where a real-time turbidimeter is available, the LAMP could also be used as a quantitative method. The LAMP assays have already been successfully developed for detection of other protozoan parasites, such as Cryptosporidium species, Trypanosoma species, Theileria species, Babesia species, Toxoplasma gondii , and Giardia duodenalis .…”

Section: Discussionmentioning

confidence: 99%

Comparative Diagnosis of Malaria Infections by Microscopy, Nested PCR, and LAMP in Northern Thailand

Pöschl¹,

Waneesorn²,

Thekisoe³

et al. 2010

The American Society of Tropical Medicine and Hygiene

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Abstract. Three methods, microscopy, nested polymerase chain reaction (nPCR), and loop-mediated isothermal amplification (LAMP) have been applied for malaria diagnosis in 105 human blood samples collected in Northern Thailand. Only Plasmodium falciparum and Plasmodium vivax infections were detected. A total number of 57 positives (54%) could be detected for P. falciparum and 25 (24%) for P. vivax when all samples that were positive in any of the three methods are counted together. The nPCR was used as a reference standard for comparison with the other methods, microscopy and LAMP. The sensitivity of LAMP for P. falciparum was 100%. All nPCR-negative samples for P. falciparum were also negative by both microscopy and LAMP (specificity, 100%). For diagnosis of P. vivax , microscopy detected 15 of 23 nPCR-positive samples (sensitivity, 65%). LAMP detected 22 of 23 nPCR-positives (sensitivity, 96%). Among the 82 nPCR-negative samples microscopy detected two samples (specificity, 98%). All 82 nPCR-negative were also negative by the LAMP method (specificity, 100%). Both Plasmodium genus-and species-specific LAMP primer sets yielded the same results in all samples. There were no significant differences in the prevalence detected by each method. We assume that LAMP was as reliable as nPCR and more reliable than microscopy in the detection of Plasmodium DNA tested in the examined Thai field blood samples. This study further validates LAMP as an alternative molecular diagnostic tool, which can be used in the diagnosis of early infections of malaria cases and together with nPCR can also be used as supplementary methods for clinical and epidemiological use.

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“…It is characterized by the use of a DNA polymerase that has low sensitivity to inhibitors and a set of four primers specially designed to recognize six different sequences on the target gene (33). Amplification occurs only when all primers bind, thus forming a product.…”

Section: Loop-mediated Isothermal Amplification (Lamp)mentioning

confidence: 99%

Molecular techniques for the study and diagnosis of parasite infection

Tavares¹,

Staggemeier²,

Borges³

et al. 2011

J. Venom. Anim. Toxins incl. Trop. Dis

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Abstract:In parasitology, routine laboratory diagnosis involves conventional methods, such as optical microscopy, used for the morphological identification of parasites. Currently, molecular biology techniques are increasingly used to diagnose parasite structures in order to enhance the identification and characterization of parasites. The objective of the present study was to review the main current and new diagnostic techniques for confirmation of parasite infections, namely: polymerase chain reaction (PCR), real-time polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), Luminex xMAP, random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and restriction fragment length polymorphism (RFLP), in addition to microsatellites. Molecular assays have comprehensively assisted in the diagnosis, treatment and epidemiological studies of parasitic diseases that affect people worldwide, helping to control parasitic disease mortality.Key words: parasite infection, diagnosis, molecular techniques, molecular epidemiology. Review ARticle The Journal of Venomous Animals and Toxins including Tropical Diseases ISSN 1678-9199 | 2011 | volume 17 | issue 3 | pages 239-248 INTRODUCTIONIn parasitology, routine laboratory diagnosis involves conventional methods, such as optical microscopy, used for the morphological identification of parasites (1). However, the occasional difficulty of identifying these parasite structures may decrease the sensitivity of such methods. Currently, because of these difficulties, molecular biology has been employed to detect parasites responsible for parasitic diseases (2).Traditional parasitological analyses have the advantage of being less costly without requiring expensive reagents and equipment. Additionally, such analyses can be easily performed when a trained microscopist is available. On the other hand, molecular technology demonstrates the presence of parasites based on their antigenic components or DNA segments (3). These tests are not influenced by environmental factors that usually can interfere with the results of a stool test, for example, thus ensuring highly reliable results (4).Current laboratory diagnostic methods for the identification of parasites include: polymerase chain reaction (PCR), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), microsatellite marker method, Luminex xMAP-based technology (areas of multianalyte profiling), loop-mediated isothermal amplification (LAMP), and the recently added real-time PCR (5-12).The objective of the present article was to review the applications of molecular biology techniques in parasitology by evaluating and discussing their uses and benefits. MOLECULAR METHODS FOR DETECTION OF PARASITE STRUCTURESSeveral molecular tests to detect parasites have been developed in the last decade. Their specificity and sensitivity have gradually increased, and parasites that were previously difficult to diagnose usi...

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Loop-Mediated Isothermal PCR (LAMP) for the Diagnosis of Falciparum Malaria

Cited by 123 publications

References 20 publications

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Comparative Diagnosis of Malaria Infections by Microscopy, Nested PCR, and LAMP in Northern Thailand

Molecular techniques for the study and diagnosis of parasite infection

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