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Detecting Phytophthora cinnamomi associated with dieback disease on Carya cathayensis using loop-mediated isothermal amplification

ABSTRACT: Chinese hickory (Carya cathayensis Sarg.) is an economically and ecologically important nut plant in China. Dieback and basal stem necrosis have been observed in the plants since 2016, and its recent spread has significantly affected plant growth and nut production. Therefore, a survey was conducted to evaluate the disease incidence at five sites in Linan County, China. The highest incidence was recorded at the Tuankou site at up to 11.39% in 2019. The oomycete, Phytophthora cinnamomi, was isolated from symptomatic plant tissue and plantation soil using baiting and selective media-based detection methods and identified. Artificial infection with the representative P. cinnamomi ST402 isolate produced vertically elongated discolorations in the outer xylem and necrotic symptoms in C. cathayensis seedlings in a greenhouse trial. Molecular detections based on loop-mediated isothermal amplification (LAMP) specific to P. cinnamomi ST402 were conducted. Result indicated that LAMP detection showed a high coherence level with the baiting assays for P. cinnamomi detection in the field. This study provides the evidence of existence of high-pathogenic P. cinnamomi in the C. cathayensis plantation soil in China and the insights into a convenient tool developed for conducting field monitoring of this aggressive pathogen.

SUBMITTER: Tong X

PROVIDER: S-EPMC8594852 | biostudies-literature |

REPOSITORIES: biostudies-literature

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An endpoint visualization loop-mediated isothermal amplification (LAMP) for detecting bubaline theileriosis.

Project description:BackgroundTropical theileriosis is a significant disease affecting the health and production levels of buffaloes in India. It is caused by an apicomplexan-Theileria annulata. The timely and accurate detection of infection is vital for implementing a mass vaccination or control programme in a given area under outbreak. Most of the literature concerned with diagnosis of theileriosis revolves around cattle, and practically, there are very limited assays available for detecting bubaline theileriosis. Loop-mediated isothermal amplification (LAMP) assay certainly amplifies the targeted deoxyribosenucleic acid (DNA) with a comparatively higher efficacy, rapidity and sensitivity. Alongside, minimal use of sophisticated instruments in performing LAMP assay is certainly an add on. The present study describes the application of LAMP assay in diagnosing tropical theileriosis in buffaloes alongside, its comparison with polymerase chain reaction (PCR) and blood microscopical examination.ResultsNo cross-reaction was seen with DNA of other haemoprotozoan. LAMP was compared with blood microscopy and PCR. LAMP detected infection in 27 out of 100 buffaloes, while blood microscopy and PCR detected disease in 16 and 24 buffaloes, respectively.ConclusionThe sensitivity, specificity and kappa value prediction of LAMP were found to be much higher than the PCR and blood microscopy. The present communication reports the first use of LAMP in detecting theileriosis in buffaloes in the world.

| S-EPMC9099057 | biostudies-literature

A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA.

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| S-EPMC5707868 | biostudies-literature

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| S-EPMC102748 | biostudies-literature

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| S-EPMC4379330 | biostudies-literature

One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV.

Project description:Due to the limitation of rapid development of specific antiviral drug or vaccine for novel emerging viruses, an accurate and rapid diagnosis is a key to manage the virus spread. We developed an efficient and rapid method with high specificity for the Middle East Respiratory Syndrome coronavirus (MERS-CoV), based on one-pot reverse transcription loop-mediated isothermal amplification (one-pot RT-LAMP). A set of six LAMP primers [F3, B3, FIP, BIP, LF (Loop-F), and LB (Loop-B)] were designed using the sequence of nucleocapsid (N) gene with optimized RT-LAMP enzyme conditions: 100 U M-MLV RTase and 4 U Bst polymerase, implying that the reaction was able to detect four infectious viral genome copies of MERS-CoV within a 60 min reaction time period. Significantly, EvaGreen dye has better signal read-out properties in one-pot RT-LAMP reaction and is more compatible with DNA polymerase than SYBR green I. Isothermally amplified specific N genes were further evaluated using field-deployable microchamber devices, leading to the specific identification of as few as 0.4 infectious viral genome copies, with no cross-reaction to the other acute respiratory disease viruses, including influenza type A (H1N1 and H3N2), type B, human coronavirus 229E, and human metapneumovirus. This sensitive, specific and feasible method provides a large-scale technical support in emergencies, and is also applied as a sample-to-detection module in Point of Care Testing devices.

| S-EPMC5220095 | biostudies-literature

Loop-mediated isothermal amplification linked a nanoparticles-based biosensor for detecting Epstein-Barr virus.

Project description:Epstein-Barr virus (EBV) is a ubiquitous gamma herpesvirus that maintains a lifelong latent association with B lymphocytes. Here, a rapid and reliable diagnosis platform for detecting EBV infection, employing loop-mediated isothermal amplification (LAMP) combined with a gold nanoparticles-based lateral flow biosensors (AuNPs-LFB) (termed LAMP Amplification Mediated AuNPs-LFB Detection, LAMAD), was developed in the current study. A set of specific LAMP primers targeting the Epstein-Barr nuclear antigen (EBNA) leader protein (EBNA-LP) gene was designed and synthesized. Subsequently, these templates extracted from various pathogens and whole blood samples were used to optimize and evaluate the EBV-LAMAD assay. As a result, the limit of detection (LoD) of the EBV-LAMAD assay was 45 copies/reaction. The EBV-LAMAD assay can detect all representative EBV pathogens used in the study, and of note, no cross-reactions were observed with other non-EBV organisms. Moreover, the whole workflow of the EBV-LAMAD assay can be completed within 70 min, including rapid EBV template preparation, EBV-LAMP amplification, and AuNPs-LFB-mediated detection. Taken together, the EBV-LAMAD assay targeting the EBNA-LP gene is a rapid, simplified, sensitive, reliable, and easy-to-use detection protocol that can be used as a competitive potential diagnostic/screening tool for EBV infection in clinical settings, especially in basic laboratories in resource-limited regions. KEY POINTS: • A novel, simplified, and easy-to-use AuNPs-LFB biosensor was designed and prepared. • LAMP combined with an AuNPs-LFB targeting the novel EBNA-LP gene was established. • EBV-LAMAD is a rapid, sensitive, and reliable detection protocol for EBV infection.

| S-EPMC10784390 | biostudies-literature

Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus.

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| S-EPMC2838873 | biostudies-literature

Development of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Detecting Feline Coronavirus.

Project description:Feline infectious peritonitis (FIP) is a worldwide fatal disease caused by a mutant feline coronavirus (FCoV). Simple and efficient molecular detection methods are needed. Here, sensitive, specific, rapid, and reliable colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect the ORF1a/1b gene of FCoV from cats with suspected FIP using neutral red as an indicator. Novel LAMP primers were specifically designed based on the gene of interest. The isothermal assay could visually detect FCoV at 58 °C for 50 min. The RT-LAMP assay was highly specific and had no cross-reactivity with other related feline viruses. The detection limit of FCoV detection by RT-LAMP was 20 fg/µL. A blind clinical test (n = 81) of the developed RT-LAMP procedure was in good agreement with the conventional PCR method. In the light of its performance specificity, sensitivity, and easy visualization, this neutral-red-based RT-LAMP approach would be a fruitful alternative molecular diagnostic tool for veterinary inspection of FCoV when combined with nucleotide sequencing or specific PCR to affirm the highly virulent FIP-associated FCoV.

| S-EPMC9405184 | biostudies-literature

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