Rapid detection of peach shoot blight caused by Phomopsis amygdali utilizing a new target gene identified from genome sequences within loop-mediated isothermal amplification

Peach shoot blight (PSB) caused by Phomopsis amygdali is a serious threat to the healthy development of the peach industry and leads to 30-50 % damage to peach production in southern China. In this study, loop-mediated isothermal amplification (LAMP) technology was used to detect the P. amygdali target of a gene of GME6801 that was unique in the whole genome of the pathogen compared with that of Diaporthe (Phomopsis) longicolla TWH P74, Fusurium graminearum PH-1, Colletotrichum gloeosporioides SMCG1 and Magnaporthe oryzae 70-15. Blast comparison of this gene sequence in NCBI database showed that no homologous sequences were found. Therefore, the gene sequence of GME6801 was used to design two pairs of LAMP primers and one pair of PCR primers. The results showed that both of primer sets were specific to the 15 strains of P. amygdali, and the other 15 fungal strains presented negative reactions, similar to the control. In addition, 50 pg of genomic DNA of P. amygdali in a 25 μl reaction system could be detected by LAMP assay which was 100 times more sensitive than PCR. Furthermore, the GME6801 LAMP assay was used to detect artificially inoculated twigs of the pathogen, disease twigs within significantly symptomatic PSB in the field and healthy twigs in the same orchard, with the detection rates of 100%, 75% and 20.8%, respectively. However, the detection rates of conventional PCR were separately 100%, 62.5% and 16.7%. The results indicated that GME6801-based LAMP could be used for P. amygdali detection as its specificity, sensitivity and simplicity. This study provides a rapid experimental basis for the identification and prediction of P. amygdali that causes PSB and is beneficial for precise prevention and control of the disease.

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Development of a rapid detection method for Trichomonas vaginalis based on loop-mediated isothermal amplification targeting adhesion protein 65

10.21203/rs.2.24375/v1 ◽

2020 ◽

Author(s):

Yuhua Li ◽

Haoran Li ◽

Xiaoxiao Song ◽

Hao Zhang ◽

Yujuan Duan ◽

...

Keyword(s):

Rapid Detection ◽

Trichomonas Vaginalis ◽

Detection Method ◽

Reaction System ◽

Lamp Assay ◽

Isothermal Amplification ◽

Detection Methods ◽

Adhesion Protein ◽

Loop Mediated Isothermal Amplification ◽

Pcr Targeting

Abstract Background: Trichomoniasis resulting from Trichomonas vaginalis (T. vaginalis) has been considered as a commonly seen disease with the transmission way of sex. At present, the detection methods of T. vaginalis mainly include wet mount microscopy, culture, PCR, immunofluorescence and ELISA. However, all of these detection methods exist shortcomings.Methods: In this study, a loop-mediated isothermal amplification (LAMP) assay that targeted the species-specific sequence of adhesion protein 65 (AP65) gene had been conducted to detect T. vaginalis. The optimum reaction system and conditions were optimized in this rapid detection method.Results: The results of sensitivity analysis showed that the LAMP assay targeting the AP65 gene was 1000 times more sensitive than the nested PCR targeting the actin gene commonly used for detection of T. vaginalis, and the detecting limitation of the former was 10 trichomonad. Moreover, the amplification of the target gene AP65 by LAMP assay exhibited high specificity and the product was exclusively from T. vaginalis. The detection technique of LAMP did not exhibit cross-reactivity with the common pathogens of Trichinella spiralis, Toxoplasma gondii, Escherichia coli, Candida albicans, Staphylococcus aureus, Haemophilus.Conclusions: According to the present study, the LAMP assay with the target of AP65 gene, was suitable for the early diagnosis of T. vaginalis infections. Consequently, the LAMP assay was proposed by the current study as a point-of-care examination and an alternative molecular tool which exhibited the potential value in the treatment, control and prevention of trichomoniasis transmission and relevant complication.

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Development of a convenient detection method for Trichomonas vaginalis based on loop-mediated isothermal amplification targeting adhesion protein 65

10.21203/rs.2.24375/v3 ◽

2020 ◽

Author(s):

Yuhua Li ◽

Shuai Wang ◽

Haoran Li ◽

Xiaoxiao Song ◽

Hao Zhang ◽

...

Keyword(s):

Trichomonas Vaginalis ◽

Detection Method ◽

Trichinella Spiralis ◽

Reaction System ◽

Lamp Assay ◽

Isothermal Amplification ◽

Detection Methods ◽

Adhesion Protein ◽

Loop Mediated Isothermal Amplification ◽

Pcr Targeting

Abstract Background: Trichomoniasis resulting from Trichomonas vaginalis (T. vaginalis) has been considered as a commonly seen disease with the transmission way of sex. At present, the detection methods of T. vaginalis mainly include wet mount microscopy, culture, PCR, immunofluorescence and ELISA. However, all of these detection methods exist shortcomings. Methods: In this study, a loop-mediated isothermal amplification (LAMP) assay that targeted the species-specific sequence of adhesion protein 65 (AP65) gene had been conducted to detect T. vaginalis. The optimum reaction system and conditions were optimized in this rapid detection method. Results: The results of sensitivity analysis showed that the LAMP assay targeting the AP65 gene was 1000 times more sensitive than the nested PCR targeting the actin gene commonly used for detection of T. vaginalis, and the detecting limitation of the former was 10 trichomonad. Moreover, the amplification of the target gene AP65 by LAMP assay exhibited high specificity and the product was exclusively from T. vaginalis. The detection technique of LAMP did not exhibit cross-reactivity with the common pathogens of Trichinella spiralis, Toxoplasma gondii, Escherichia coli, Candida albicans, Staphylococcus aureus, Haemophilus. Conclusions: According to the present study, the LAMP assay with the target of AP65 gene, was suitable for the early diagnosis of T. vaginalis infections. Consequently, the LAMP assay was proposed by the current study as a point-of-care examination and an alternative molecular tool which exhibited the potential value in the treatment, control and prevention of trichomoniasis transmission and relevant complication.

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Rapid Visualization and Detection of Staphylococcus Aureus Based on Loop-Mediated Isothermal Amplification

10.21203/rs.3.rs-763986/v1 ◽

2021 ◽

Author(s):

Chuan Wu ◽

Yuanyuan Zeng ◽

Yang He

Keyword(s):

Staphylococcus Aureus ◽

Limit Of Detection ◽

Reaction System ◽

Bacterial Pathogen ◽

Lamp Assay ◽

Cross Reactivity ◽

Isothermal Amplification ◽

Diagnostic Efficacy ◽

Diverse Range ◽

Loop Mediated Isothermal Amplification

Abstract Staphylococcus aureus is a common clinical bacterial pathogen that can cause a diverse range of infections. The establishment of a rapid and reliable assay for the early diagnosis and detection of S. aureus is of great significance. In this study, we developed a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus using the colorimetric indicator hydroxy naphthol blue (HNB). The LAMP reaction was optimized by adjusting the amplification temperature, the concentrations of Mg2+, dNTP, and HNB, and the incubation time. In the optimized reaction system, the specificity of LAMP for S. aureus was 100%. The results established that this method accurately identified S. aureus, with no cross-reactivity with 16 non-S. aureus strains. The limit of detection (LOD) of LAMP was 8 copies/reaction of purified plasmid DNA or 400 colony-forming units/reaction of S. aureus. Compared with conventional PCR, LAMP lowered the LOD by 10-fold. Finally, 220 clinically isolated strains of S. aureus and 149 non-S. aureus strains were used to evaluate the diagnostic efficacy of LAMP. The findings indicated that LAMP is a reliable test for S. aureus and could be a promising tool for the rapid diagnosis of S. aureus infections.

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Real-time loop-mediated isothermal amplification (LAMP) of mgc2 gene of Mycoplasma gallisepticum

Journal of Veterinary Research ◽

10.1515/jvetres-2017-0058 ◽

2017 ◽

Vol 61 (4) ◽

pp. 439-444 ◽

Cited By ~ 1

Author(s):

Syed Ehtisham-ul-Haque ◽

Madiha Kiran ◽

Usman Waheed ◽

Muhammad Younus

Keyword(s):

Real Time ◽

Gene Sequence ◽

Molecular Detection ◽

Lamp Assay ◽

Mycoplasma Gallisepticum ◽

Isothermal Amplification ◽

Economic Losses ◽

Poultry Industry ◽

Loop Mediated Isothermal Amplification ◽

Time Loop

AbstractIntroduction:Mycoplasma gallisepticum is considered the most pathogenic and economically significant avian Mycoplasma spp. for the worldwide poultry industry. The aim of this study was to develop a novel and sensitive real-time loop-mediated isothermal amplification (LAMP) assay based on the amplification of its mgc2 gene sequence for its rapid molecular detection in poultry.Material and Methods: Blood samples from 300 broiler and layer chickens were screened using a rapid serum agglutination (RSA) test. A real-time LAMP reaction was conducted with seropositive swab samples at 60°C for 90 min in an ESEQuant tube scanner using 6-carboxyfluorescein as the reporting dye.Results: The sensitivity of the developed assay was 10 fg/μL of DNA. The assay was found 100% specific, showing no cross-reactivity with other avian Mycoplasma species. The proportion found of the positive samples by the real-time LAMP was 58%. In comparison, the RSA was found to detect 52% of positive cases.Conclusion: The mgc2 real-time LAMP emerged as a more sensitive and accurate method for molecular detection of M. gallisepticum than RSA. Robustness and precision give it applicability as a potential field diagnostic tool for M. gallisepticum control. The study will be beneficial in reducing economic losses that M. gallisepticum inflicts on the poultry industry. This is the first reported development of a real-time LAMP assay based on the amplification of the mgc2 gene sequence using an ESEQuant tube scanner for galline M. gallisepticum detection.

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Detection of Colletotrichum acutatum Sensu Lato on Strawberry by Loop-Mediated Isothermal Amplification

Plant Disease ◽

10.1094/pdis-09-15-1013-re ◽

2016 ◽

Vol 100 (9) ◽

pp. 1804-1812 ◽

Cited By ~ 12

Author(s):

X. Zhang ◽

T. C. Harrington ◽

J. C. Batzer ◽

R. Kubota ◽

N. A. Peres ◽

...

Keyword(s):

Pure Culture ◽

Detection Threshold ◽

Lamp Assay ◽

Its Region ◽

Isothermal Amplification ◽

Fruit Rot ◽

Colletotrichum Acutatum ◽

Initial Inoculum ◽

Loop Mediated Isothermal Amplification ◽

Primer Sets

Colletotrichum acutatum, one of the most economically damaging pathogens of strawberry, is the primary causal agent of anthracnose fruit rot (AFR). A key challenge in managing AFR is detecting the pathogen on asymptomatic plants. To meet this need, a loop-mediated isothermal amplification (LAMP) assay was developed that incorporated two sets of primers: LITSG1, targeted on the intergenic transcribed spacer (ITS) region of ribosomal DNA, and Ltub2, on the β-tubulin 2 gene. In pure culture assays, Ltub2 was specific for detection of C. acutatum, whereas LITSG1 detected C. acutatum and two additional anthracnose pathogens, C. gloeosporioides and C. fragariae. LITSG1 had 10-fold lower detection threshold (20 pg of mycelial DNA) than Ltub2 (200 pg mycelial DNA) in detection of C. acutatum from pure culture. For detection on asymptomatic leaves, two protocols for dislodging C. acutatum for DNA extraction were compared: i) the sonicate-agitate (SA) method and ii) the freeze-incubate-sonicate-agitate (FISA) method, which initially freezes tissues, followed by 2 days of incubation at 26°C in darkness, and then, sonication and agitation. Both methods were used for greenhouse-grown plant leaves that had been spray inoculated with serial dilutions ranging from 1.5 × 106 to 1.5 conidia ml−1. The FISA method produced more repeatable results than the SA method. For the FISA method, detection limits (expressed as initial inoculum concentrations) using LITSG1 and Ltub2 were 1.5 × 101 and 1.5 × 102 conidia ml−1, respectively. For composite samples comprised of inoculated (1.5 × 106 conidia ml−1) and noninoculated leaves of greenhouse-grown strawberry, the two sets of LAMP primers were compared using the SA method. Primer set LITSG1 consistently detected the pathogen from a single inoculated leaf in bulk samples of 50 or fewer pathogen-free leaves, whereas Ltub2 consistently detected one inoculated leaf in 20 or fewer pathogen-free leaves. Using primer set LITSG1, FISA was more sensitive than SA for detecting C. acutatum on leaves of field-grown plants from Florida. In an Iowa field trial using the FISA method, both primer sets detected C. acutatum in samples of asymptomatic leaves 6 days before fruit symptoms appeared. The results indicate that the LAMP assay has potential to provide a simplified method for detection of C. acutatum on asymptomatic strawberry plants.

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Loop-mediated isothermal amplification for diagnosing marine pathogens in tissues of Crassostrea spp. and white shrimp, Litopenaeus vannamei, farmed in Mexico

Ciencias Marinas ◽

10.7773/cm.v47i4.3095 ◽

2021 ◽

Vol 47 (4) ◽

Author(s):

Ismael Mendoza-Avilés ◽

Carla A Muñoz-Rojas ◽

Mario Rojas ◽

Norma Estrada

Keyword(s):

Disease Transmission ◽

Target Genes ◽

Reaction Times ◽

Lamp Assay ◽

Isothermal Amplification ◽

White Shrimp ◽

Loop Mediated Isothermal Amplification ◽

Aquaculture Industry ◽

Candidate Target ◽

Primer Sets

Loop-mediated isothermal amplification (LAMP) is an accurate, sensitive, rapid, and easy-to-perform method for gene amplification under isothermal conditions, and it has served as a powerful diagnostic tool. In this study, we used LAMP to develop a diagnostic protocol for detecting Vibrio parahaemolyticus and white spot syndrome virus in whiteleg shrimp, and Perkinsus spp. in Crassostrea spp. in Mexico. These pathogens are associated with different diseases and are considered a threat in the aquaculture industry. Infected and uninfected oysters and shrimp were obtained from farms in the northwest coast of Mexico to standardize the LAMP assay. We determined the candidate target genes in the first-round analysis of many sets of primers, and then we chose a set of primers that successfully amplified with Mexican samples. We optimized the LAMP reactions for each pathogen with the chosen primer sets using temperature gradients from 61 to 65 ºC, DNA concentrations from 2.5 pg to 250.0 ng, and reaction times from 10 to 60 min. This study established a diagnostic procedure for detecting pathogens in oysters and shrimp from Mexico. Early diagnosis and treatment of pathogens can immensely reduce disease transmission in aquaculture farms.

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Development of a multiplex Loop-Mediated Isothermal Amplification (LAMP) assay for on-site diagnosis of SARS CoV-2

PLoS ONE ◽

10.1371/journal.pone.0248042 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0248042

Author(s):

Woong Sik Jang ◽

Da Hye Lim ◽

Jung Yoon ◽

Ahran Kim ◽

Minsup Lim ◽

...

Keyword(s):

South Korea ◽

Real Time ◽

Real Time Pcr ◽

Detection Limits ◽

Lamp Assay ◽

Isothermal Amplification ◽

Clinical Samples ◽

Lamp Primer ◽

Loop Mediated Isothermal Amplification ◽

Primer Sets

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).

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Molecular Detection of QoI Resistance in Colletotrichum gloeosporioides Causing Strawberry Anthracnose Based on Loop-Mediated Isothermal Amplification Assay

Plant Disease ◽

10.1094/pdis-09-18-1593-re ◽

2019 ◽

Vol 103 (6) ◽

pp. 1319-1325 ◽

Cited By ~ 1

Author(s):

J. Y. Wu ◽

X. R. Hu ◽

C. Q. Zhang

Keyword(s):

Colletotrichum Gloeosporioides ◽

Lamp Assay ◽

Practical Method ◽

Conventional Polymerase Chain Reaction ◽

Isothermal Amplification ◽

Time Saving ◽

Loop Mediated Isothermal Amplification ◽

Quinone Outside Inhibitor ◽

Strawberry Anthracnose ◽

Primer Sets

Anthracnose is one of the most common diseases in strawberry plants. Colletotrichum gloeosporioides is the major cause of anthracnose in China, including Zhejiang Province. Early, specific, reliable, and time-saving detection is urgently needed to prevent the further spread of C. gloeosporioides, guiding farmers to utilize chemicals to control anthracnose. In this study, we showed that the high resistance to pyraclostrobin, caused by a point mutation at codon 143 (GGT→GCT) in the cytochrome b gene of C. gloeosporioides was prevalent in the strawberry growing regions, and we developed a loop-mediated isothermal amplification (LAMP) assay as a detection method. Primer sets S0 and S4 could be used to specifically detect C. gloeosporioides isolates and the G143A mutations, respectively. A detection limit of 10−2 ng (10 pg), which is at least 10-fold more sensitive than conventional polymerase chain reaction, was achieved by the LAMP assay. Here, we utilized lateral-flow devices (LFDs), nitrocellulose membranes that can absorb nucleic acids, to acquire the total genomic DNA of strawberry plants within 2 min. The LFD membranes were used as DNA templates for the LAMP assays to accurately detect strawberry plants infected with C. gloeosporioides. This diagnostic method for strawberry anthracnose was accomplished within 1 h, including the sample preparation and LAMP assays. Collectively, we developed a sensitive and practical method for monitoring C. gloeosporioides and its quinone outside inhibitor–resistant mutants. The LAMP assay for detection of C. gloeosporioides in strawberry plants has great potential for rapid strawberry anthracnose surveillance and will provide farmers with advice on preventing C gloeosporioides at the early stages of strawberry development.

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Development of a convenient detection method for Trichomonas vaginalis based on loop-mediated isothermal amplification targeting adhesion protein 65

10.21203/rs.2.24375/v2 ◽

2020 ◽

Author(s):

Yuhua Li ◽

Shuai Wang ◽

Haoran Li ◽

Xiaoxiao Song ◽

Hao Zhang ◽

...

Keyword(s):

Trichomonas Vaginalis ◽

Detection Method ◽

Trichinella Spiralis ◽

Reaction System ◽

Lamp Assay ◽

Isothermal Amplification ◽

Detection Methods ◽

Adhesion Protein ◽

Loop Mediated Isothermal Amplification ◽

Pcr Targeting

Abstract Background: Trichomoniasis resulting from Trichomonas vaginalis ( T. vaginalis ) has been considered as a commonly seen disease with the transmission way of sex. At present, the detection methods of T. vaginalis mainly include wet mount microscopy, culture, PCR, immunofluorescence and ELISA. However, all of these detection methods exist shortcomings. Methods: In this study, a loop-mediated isothermal amplification (LAMP) assay that targeted the species-specific sequence of adhesion protein 65 (AP65) gene had been conducted to detect T. vaginalis . The optimum reaction system and conditions were optimized in this rapid detection method. Results: The results of sensitivity analysis showed that the LAMP assay targeting the AP65 gene was 1000 times more sensitive than the nested PCR targeting the actin gene commonly used for detection of T. vaginalis , and the detecting limitation of the former was 10 trichomonad. Moreover, the amplification of the target gene AP65 by LAMP assay exhibited high specificity and the product was exclusively from T. vaginalis . The detection technique of LAMP did not exhibit cross-reactivity with the common pathogens of Trichinella spiralis , Toxoplasma gondii , Escherichia coli , Candida albicans , Staphylococcus aureus , Haemophilus . Conclusions: According to the present study, the LAMP assay with the target of AP65 gene, was suitable for the early diagnosis of T. vaginalis infections. Consequently, the LAMP assay was proposed by the current study as a point-of-care examination and an alternative molecular tool which exhibited the potential value in the treatment, control and prevention of trichomoniasis transmission and relevant complication.

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