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Brown root rot caused by Phytopythium vexans is a new destructive root disease on many plants such as Gingko, Citrus, kiwifruit, and ramie. The establishment of loop-mediated isothermal amplification (LAMP) technology for detecting P. vexans can help monitor and control brown root rot quickly, efficiently, and accurately. LAMP technology is known for its simplicity, sensitivity, and speed; and it does not require any specialized equipment – a water bath or a thermoblock is sufficient for isothermal amplifications. LAMP products can be visualized by using hydroxy naphthol blue (HNB) dye or agarose gel electrophoresis. In this study, by searching and comparing the internal transcribed spacer (ITS) sequences of P. vexans and the related species in oomycete genera Pythium, Phytopythium, and Phytophthora, we designed specific primers targeting the ITS gene region of P. vexans. Using HNB dye, we established a LAMP technique for rapid detection of P. vexans by visible color change. In addition, we optimized the protocol to enhance both sensitivity and specificity for P. vexans detection. Under the optimized condition, our protocol based on LAMP technology could detect as low as 24 copies of the P. vexans genomic DNA, which is ∼100 times more sensitive than conventional PCR. This method can successfully detect P. vexans using cell suspensions from P. vexans – infected ramie root tissues.

Brown root rot caused by Phytopythium vexans is a new destructive root disease on many plants such as Gingko, Citrus, kiwifruit, and ramie. The establishment of loop-mediated isothermal amplification (LAMP) technology for detecting P. vexans can help monitor and control brown root rot quickly, efficiently, and accurately. LAMP technology is known for its simplicity, sensitivity, and speed; and it does not require any specialized equipment – a water bath or a thermoblock is sufficient for isothermal amplifications. LAMP products can be visualized by using hydroxy naphthol blue (HNB) dye or agarose gel electrophoresis. In this study, by searching and comparing the internal transcribed spacer (ITS) sequences of P. vexans and the related species in oomycete genera Pythium, Phytopythium , and Phytophthora , we designed specific primers targeting the ITS gene region of P. vexans. Using HNB dye, we established a LAMP technique for rapid detection of P. vexans by visible color change. In addition, we optimized the protocol to enhance both sensitivity and specificity for P. vexans detection. Under the optimized condition, our protocol based on LAMP technology could detect as low as 24 copies of the P. vexans genomic DNA, which is ∼100 times more sensitive than conventional PCR. This method can successfully detect P. vexans using cell suspensions from P. vexans – infected ramie root tissues. [ABSTRACT FROM AUTHOR]

Fire blight remains a serious threat to commercial apple production in the USA and worldwide. Other diseases and spray damage can result in fire blight-like symptoms that can lead to misdiagnosis and affect disease management strategies. Accurate and timely detection of the fire blight pathogen, Erwinia amylovora, is extremely important to deploy appropriate and timely measures to reduce fire blight epidemics in commercial apple orchards. We tested two commercial lateral flow immunoassays (AgriStrip®, and Pocket Diagnostics kit), Loop mediated isothermal amplification (LAMP), and quantitative PCR (qPCR) to diagnose E. amylovora infected samples in lab and field settings. The AgriStrip® and Pocket Diagnostics kits were able to detect actively growing bacteria up to ×106 cfu/ml bacterial concentration. Pocket Diagnostics kit had less specificity and showed positive tests for E. pyrifolia in addition to E. amylovora. The LAMP assay showed high specificity for E. amylovora and was able to detect up to ×103 cfu/ml bacterial concentrations. The qPCR assay was also able to detect bacterial cells up to ×10−3 cfu/ml bacterial concentration with highly specific E. amylovora detection. Grower surveys and comparative cost-benefit analysis indicated that immunoassay kits are less expensive, easier to use, and require less technical expertise for on-site fire blight diagnosis than LAMP and qPCR. However, the choice of a specific diagnostic assay depends on the time, sensitivity, and specificity required for the detection of fire blight and its management. [ABSTRACT FROM AUTHOR]

Introduction: Though decades back the fungi were considered as inconsequential causes of infection but in recent years there has been a surge. This could be due to AIDS epidemic or because of advances in the medical care and treatment. A number of diagnostic procedures are being used but each has its own limitations. Accurate diagnosis relies precisely on a combination of microbiological, histopathological and serological evidence. Aims: This article focuses on specific conventional and advanced techniques concerning a practical approach to the laboratory investigations of fungal infections. Methods: Data was obtained and analyzed from previously published literature and electronic database searches from PubMed and Google Scholar. Results: Traditional methods of diagnosis include direct microscopic examination of samples, culture, serology and histopathology. Nowadays with advances in diagnostic methods, molecular diagnostics and antigen detection in clinical samples is being highly recommended. Conclusion: Although, the number of species that are routinely seen causing infection is quite low, yet new species are continually being implicated. This has forced the investigators to develop molecular methods for fungal identification. These methods have and will continue to have a major impact on the diagnosis and appropriate treatment of fungal infection. Analytical parameters of these methods need to be standardized to optimize sensitivity and specificity and comparative studies need to be performed to determine which are best to use in the laboratory. [ABSTRACT FROM AUTHOR]

Abstract There is need for rapid and cost-effective diagnostic test for tuberculosis. The present study was carried out to design a Loop-mediated isothermal amplification (LAMP) assay combined with lateral flow dipstick (LFD) as a point-of-care method for diagnosis of TB. LAMP assay targeting sdaA gene combined with LFD for sequence specific detection was standardized in user friendly and rapid format. It does not require sophisticated instruments and shows visual results instantly. The LAMP-LFD assay was validated using culture confirmed specimens. The assay was evaluated in a cross-sectional study using respiratory specimens collected from patients in Delhi, India and it showed high concordance with GeneXpert MTB/RIF assay. Lateral flow dipstick method has provided an excellent detection format with LAMP method. The LAMP-LFD assay showed high diagnostic accuracy in comparison to other methods and can be used as a point-of-care test in cost-effective manner. Graphical abstract Unlabelled Image Highlights • Rapid and sensitive detection of Mycobacterium tuberculosis. • No need for sophisticated instruments. • Naked eye visualization of results using Lateral flow dipstick. • Sequence specific method for high specificity. • Potential point of care diagnostic test for resource limited settings. [ABSTRACT FROM AUTHOR]

Introduction: The loop-mediated isothermal amplification (LAMP) technique holds substantial promise as an alternative easy-to-use molecular test for malaria parasite detection. Several modifications to the initial malaria LAMP assay have been made in an effort to make the LAMP platform more field-friendly. Areas covered: A PubMed literature search was performed using the following search terms: ‘malaria,’ ‘loop mediated isothermal amplification’, ‘LAMP’, ‘molecular tests’ and ‘diagnostics’. The authors review the currently reported malaria LAMP assays and discuss what requirements would be needed to make malaria LAMP assays field-usable, especially in the context of malaria elimination. Expert commentary: Expanding the malaria LAMP tests as options for use in malaria control programs will require addressing some important challenges such as the need for simplified sample preparation steps; ready to use kits that require no cold chain; the use of a non-subjective results readout and preferably cost-effectiveness. Two malaria LAMP kits are now CE-marked and commercially available: the Loopamp MALARIA kit and theIllumigenemalaria LAMP. Malaria LAMP tests, like other molecular tests, will likely be utilized in very specific studies such as: to evaluate ‘detect and treat’ strategies; in controlled malaria infection trials or drug efficacy trials and as confirmatory test in reference laboratories. [ABSTRACT FROM PUBLISHER]

In the present study, IS 6110 loop mediated isothermal amplification (LAMP) assay was modified using dUTP-UNG (uracil-DNA N -glycosylase) strategy to prevent carryover contamination, and was evaluated using clinical specimens. The clinical specimens were collected from 236 suspected patients of pulmonary tuberculosis and 315 specimens of suspected patients of extra pulmonary tuberculosis. DNA was extracted from specimens and used as template for nucleic acid amplification. The results were evaluated with culture method as gold standard. Modified IS 6110 LAMP assay showed high sensitivity (94.4%) and specificity (97.2%) in specimens collected from suspected pulmonary tuberculosis patients. Sensitivity was comparatively less (86.67%) in extra pulmonary specimens while specificity was 94.04%. In conclusion, IS 6110 LAMP assay was modified to prevent carry over contamination and it was validated to be rapid, sensitive and specific method with prospective application in resource-limited settings. [ABSTRACT FROM AUTHOR]

Mycoplasma genitalium is a sexually transmissible, pathogenic bacterium and a significant cause of nongonococcal urethritis in both men and women. Due to the difficulty of the culture of M. genitalium from clinical samples, the laboratory diagnosis of M. genitalium infection is almost exclusively carried out using nucleic acid amplification tests. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology, utilising a set of 4 primers specific to 6 distinct regions of the target DNA sequence, in order to amplify target DNA in a highly specific and rapid manner. A LAMP assay was designed to the pdhD gene of M. genitalium , and the limit of detection of the assay was determined as 10 fg of M. genitalium genomic DNA, equating to ~16 copies of the M. genitalium genome, which was equally sensitive as a gold standard 16S rRNA polymerase chain reaction assay. [ABSTRACT FROM AUTHOR]

Background: To detect all malaria infections in elimination settings sensitive, high throughput and field deployable diagnostic tools are required. Loop-mediated isothermal amplification (LAMP) represents a possible field-applicable molecular diagnostic tool. However, current LAMP platforms are limited by their capacity for high throughput. Methods: A high-throughput LAMP (HtLAMP) platform amplifying mitochondrial targets using a 96-well microtitre plate platform, processing 85 samples and 11 controls, using hydroxynaphtholblue as a colourimetric indicator was optimized for the detection of malaria parasites. Objective confirmation of visually detectable colour change results was made using a spectrophotometer. A dilution series of laboratory-cultured 3D7 Plasmodium falciparum parasites was used to determine the limit of detection of the HtLAMP assay, using P. falciparum (HtLAMP-Pf) and Plasmodium genus (HtLAMP-Pg) primers, on whole blood and filter paper, and using different DNA extraction protocols. The diagnostic accuracy of HtLAMP was validated using clinical samples from Papua New Guinea, Malaysia, Ghana and The Gambia and its field applicability was evaluated in Kota Marudu district hospital, Sabah, Malaysia. Results: The HtLAMP assay proved to be a simple method generating a visually-detectable blue and purple colour change that could be objectively confirmed in a spectrophotometer at a wavelength of 600 nm. When compared with PCR, overall HtLAMP-Pg had a sensitivity of 98 % (n = 260/266, 95 % Cl 95-99) and specificity 83 % (n = 15/18, 95 % Cl 59-96). HtLAMP-Pf had a sensitivity of 97 % (n = 124/128, 95 % Cl 92-99) and specificity of 96 % (n = 151/157, 95 % Cl 92-99). A validation study in a regional hospital laboratory demonstrated ease of performance and interpretation of the HtLAMP assay. HtLAMP-Pf performed in this field setting had a sensitivity of 100 % (n = 17/17, 95 % Cl 80-100) and specificity of 95 % (n = 123/128,95 % Cl 90-98) compared with multiplex PCR. HtLAMP-Pf also performed well on filter paper samples from asymptomatic Ghanaian children with a sensitivity of 88 % (n = 23/25, 95 % Cl 69-97). Conclusion: This colourimetric HtLAMP assay holds much promise as a field applicable molecular diagnostic tool for the purpose of malaria elimination. [ABSTRACT FROM AUTHOR]

Abstract Background Buruli ulcer (BU), a neglected tropical skin disease caused by Mycobacterium ulcerans, has been reported in over 30 countries worldwide and is highly endemic in rural West and Central Africa. The mode of transmission remains unknown and treatment is the only alternative to disease control. Early and effective treatment to prevent the morbid effects of the disease depends on early diagnosis; however, current diagnosis based on clinical presentation and microscopy has to be confirmed by PCR and other tests in reference laboratories. As such confirmed BU diagnosis is either late, inefficient, time consuming or very expensive, and there is the need for an early diagnosis tool at point of care facilities. In this paper we report on a simple, quick and inexpensive diagnostic test that could be used at point of care facilities, in resource-poor settings. Methods The methodology employed is based on the loop mediated isothermal amplification (LAMP) technique. Four sets of Primers, targeting the mycolactone encoding plasmid genome sequence of M. ulcerans were designed. The BU-LAMP assay was developed and tested on five M. ulcerans strains from patients in Ghana and two American Type Culture Control (ATCC) reference isolates; Ghana #970321 (D19F9) and Benin #990826 (D27D14). We also tested the assay on other closely related, mycolactone-producing mycobacterial strains; M. marinum 1218, M. marinum DL240490, M. liflandii and M. pseudoshotsii, as well as experimentally infected laboratory animal and clinical samples. Results The results revealed a high specificity of the BU-LAMP assay for selectively detecting M. ulcerans. Compared to the conventional IS-2404 PCR, the new assay is cheaper and simpler and ten times more sensitive. Test results can be obtained within 1 hour. Conclusions This study indicates that the BU-LAMP assay could be suitable for early disease diagnosis and application in low-resource health facilities.

Vector-borne diseases are rising in interest due to global warming, which is believed to impact on the distribution of vectors into new areas thus influencing the occurrence and epidemiology of vector-borne pathogens. Babesia canis belongs to the Piroplasmidae and there are three described subspecies, namely B. canis canis, B. canis rossi and B. canis vogeli. They are each transmitted by a different tick-species, Dermacentor reticulatus, Haemaphysalis leachi and Rhipicephalus sanguineus, respectively. There are also differences in the geographical distribution and pathogenicity to dogs of each subspecies. In this study, we aimed to establish a rapid and easy to perform DNA-based test using loop-mediated isothermal amplification to detect all three Babesia canis subspecies in one assay. [ABSTRACT FROM AUTHOR]