scholarly journals Characteristics of Isolates of Pseudomonas aeruginosa and Serratia marcescens Associated With Post-harvest Fuzi (Aconitum carmichaelii) Rot and Their Novel Loop-Mediated Isothermal Amplification Detection Methods

2021 ◽  
Vol 12 ◽  
Author(s):  
Meng Fu ◽  
Xin Zhang ◽  
Bei Chen ◽  
Mingzhu Li ◽  
Guoyan Zhang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Serratia Marcescens ◽  
Target Genes ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Unknown Etiology ◽  
Bacterial Strains ◽  
Post Harvest ◽  
Loop Mediated Isothermal Amplification ◽  
Aconitum Carmichaelii

Fuzi (the lateral root of Aconitum carmichaelii Debx.) is a traditional Chinese medicine that is cultivated in more than eight provinces in China. However, it can be easily devastated by post-harvest rot, causing huge losses. Therefore, it is extremely important that the primary causal pathogens of post-harvest Fuzi rot are identified and appropriate detection methods for them are developed to prevent and control losses. In this study, two bacterial strains (X1 and X2) were isolated from rotten post-harvest Fuzi. Based on their morphological, physiological, and biochemical characteristics, housekeeping gene homologies, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS) results, these isolates were identified as Pseudomonas aeruginosa and Serratia marcescens. The pathogenicities of these isolates were confirmed by fulfilling Koch’s postulates demonstrating that they were post-harvest Fuzi rot pathogens. Two loop-mediated isothermal amplification (LAMP) methods targeting the gyrase B subunit (gyrB) gene of P. aeruginosa and the phosphatidylinositol glycan C (pigC) gene of S. marcescens were successfully developed, and it was found that the target genes were highly specific to the two pathogens. These LAMP methods were used to detect P. aeruginosa and S. marcescens in 46 naturally occurring Fuzi and their associated rhizosphere soil samples of unknown etiology. The two bacterial assays were positive in some healthy and rotten samples and could be accomplished within 1 h at 65°C without the need for complicated, expensive instruments. To our knowledge, this is the first report of P. aeruginosa and S. marcescens causing post-harvest Fuzi rot. The newly developed methods are expected to have applications in point-of-care testing for the two pathogens under different Fuzi planting procedures and will significantly contribute to the control and prevention of Fuzi rot.

scholarly journals A Portable Automatic Endpoint Detection System for Amplicons of Loop Mediated Isothermal Amplification on Microfluidic Compact Disk Platform

Sensors ◽  
2015 ◽  
Vol 15 (3) ◽  
pp. 5376-5389 ◽  
Author(s):  
Shah Uddin ◽  
Fatimah Ibrahim ◽  
Abkar Sayad ◽  
Aung Thiha ◽  
Koh Pei ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Detection System ◽  
Liquid Crystal Display ◽  
Foodborne Pathogen ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Endpoint Detection ◽  
Compact Disk ◽  
Loop Mediated Isothermal Amplification ◽  
The Impact

In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10−3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment.

scholarly journals Development of a Loop-mediated isothermal amplification (LAMP) technique for specific and early detection of Mycobacterium leprae in clinical samples

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nupur Garg ◽  
Upasana Sahu ◽  
Sudeshna Kar ◽  
Farhan J. Ahmad
Keyword(s):  
Early Detection ◽  
Mycobacterium Leprae ◽  
Negative Control ◽  
Rapid Diagnosis ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Loop Mediated Isothermal Amplification

AbstractLeprosy, a progressive, mutilating and highly stigmatized disease caused by Mycobacterium leprae (ML), continues to prevail in the developing world. This is due to the absence of rapid, specific and sensitive diagnostic tools for its early detection since the disease gets notified only with the advent of physical scarring in patients. This study reports the development of a Loop-mediated isothermal amplification (LAMP) technique for fast, sensitive and specific amplification of 16S rRNA gene of ML DNA for early detection of leprosy in resource-limited areas. Various parameters were optimized to obtain robust and reliable amplification of ML DNA. Blind clinical validation studies were performed which showed that this technique had complete concurrence with conventional techniques. Total absence of amplification of negative control DNA confirmed the specificity of this test. Various visual detection methods viz. colorimetric, turbidity differentiation and bridge flocculation were standardized to establish easy-to-read and rapid diagnosis. This technique eliminates the lack of accuracy and sensitivity in skin smear tests in patients and the requirement for expensive lab equipments and trained technicians. The technique holds promise for further expansion and has the potential to cater to the unmet needs of society for a cheap, highly-sensitive and robust rapid diagnosis of ML.

HIGHLY SENSITIVE H7N9 AVIAN INFLUENZA VIRUS DETECTION USING REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION AND OLIGONUCLEOTIDE MICROARRAY

2015 ◽  
Vol 41 (04) ◽  
pp. 251-255
Author(s):  
Lih-Chiann Wang ◽  
Dean Huang
Keyword(s):  
Avian Influenza ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
High Sensitivity ◽  
Oligonucleotide Microarray ◽  
Influenza Viruses ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Loop Mediated Isothermal Amplification ◽  
Viral Copy Number

H7N9 avian influenza viruses have circulated in the human population and poultry flocks in China since 2013. H7N9 virus monitoring is imperative in Taiwan due to the frequent contact between China and Taiwan. Traditional viral molecular detection methods using RT-PCR and sequencing are time- and labor-intensive, thus a simpler and cheaper method with high sensitivity and specificity is worth developing. We successfully detected human and wild bird H7N9 viruses in this study using reverse transcription loop-mediated isothermal amplification (RT-LAMP) and oligonucleotide microarray. The detection limit was as low as one viral copy number. The specific matching reaction between the templates and microarray probes during hybridization ensured the high detection effectiveness. The excellent sensitivity and specificity of the RT-LAMP-microarray makes it a powerful H7N9 surveillance approach in Taiwan.

scholarly journals Development of a Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of the tdh and trh Genes of Vibrio parahaemolyticus and Related Vibrio Species

2009 ◽  
Vol 76 (3) ◽  
pp. 820-828 ◽  
Author(s):  
Wataru Yamazaki ◽  
Yuko Kumeda ◽  
Naoaki Misawa ◽  
Yoshitsugu Nakaguchi ◽  
Mitsuaki Nishibuchi
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Virulence Determinants ◽  
Bacterial Strains ◽  
Phenotypic Differences ◽  
Loop Mediated Isothermal Amplification ◽  
Vibrio Mimicus ◽  
Thermostable Direct Hemolysin

ABSTRACT Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.

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

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.

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

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.

scholarly journals Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Method for the Detection of Salmonella spp. in Terms of Sensitivity and Applicability

2020 ◽  
pp. 1-5
Author(s):  
Yanhong Liu ◽  
Deguo Wang ◽  
Meng Zhang ◽  
Yanhong Liu ◽  
Yongzhen Wang
Keyword(s):  
Genomic Dna ◽  
Pathogenic Bacteria ◽  
Detection Limits ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
23S Rrna ◽  
Lamp Method ◽  
Salmonella Spp ◽  
Loop Mediated Isothermal Amplification ◽  
Food Borne

Salmonella spp. are important food-borne pathogens that can cause diseases in humans. Many detection methods have been established in Salmonella spp. using loop-mediated isothermal amplification (LAMP) or reverse transcription loop-mediated isothermal amplification (RT-LAMP). The detection limits of these assays varied from 1 CFU/reaction to 104 CFU/reaction, from 100 fg genomic DNA/reaction to 10 pg genomic DNA/reaction, or from 2.0×101 CFU/mL to 107 CFU/mL for food samples. In this study, LAMP assays were developed using genomic DNA for the detection of Salmonella spp. Two sets of LAMP primers were designed using the invA gene and the 16S-23S rRNA intergenic spacer region (ITS) of S. enterica as the target sequences for two LAMP assays. The detection limits of the two methods were respectively 20 pg S. enterica DNA/reaction and 10 pg S. enterica DNA/reaction at the optimized temperature, and the LAMP methods were of high repeatability and specificity for S. enterica detection. This study provides a baseline for the application of LAMP for the detection of food-borne pathogenic bacteria.

scholarly journals Evaluation of the real-time fluorescence loop-mediated isothermal amplification assay for the detection of Streptococcus agalactiae

2019 ◽  
Vol 39 (5) ◽  
Author(s):  
Xu-Guang Guo ◽  
Ya-Ru Zhuang ◽  
Jin-Zhou Wen ◽  
Tian-Ao Xie ◽  
Ye-Ling Liu ◽  
...  
Keyword(s):  
Real Time ◽  
Streptococcus Agalactiae ◽  
Rapid Detection ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Bacterial Strains ◽  
Loop Mediated Isothermal Amplification ◽  
Dna Strand Displacement ◽  
Time Amplification

Abstract Streptococcus agalactiae is a major pathogenic bacterium causing perinatal infections in humans. In the present study, a novel real-time fluorescence loop-mediated isothermal amplification technology was successfully developed and evaluated for the detection of S. agalactiae in a single reaction. Six specific primers were designed to amplify the corresponding six regions of fbs B gene of S. agalactiae, using Bst DNA polymerase with DNA strand displacement activity at a constant temperature for 60 min. The presence of S. agalactiae was indicated by the fluorescence in real-time. Amplification of the targeted gene fragment was optimized with the primer 1 in the current setup. Positive result was only obtained for Sa by Real-LAMP among 10 tested relevant bacterial strains, with the detection sensitivity of 300 pg/µl. Real-LAMP was demonstrated to be a simple and rapid detection tool for S. agalactiae with high specificity and stability, which ensures its wide application and broad prospective utilization in clinical practice for the rapid detection of S. agalactiae.

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