Highly Sensitive Loop-Mediated Isothermal Amplification for the Detection of Leptospira

Leptospirosis is a worldwide zoonosis caused by an infection with the pathogenic species of Leptospira. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the DNA of Leptospira spp. Six sets of primers targeting the gene of the subsurface protein, lipL32, were evaluated for their detection sensitivity. The best primer set detected less than 25 copies of lipL32 per reaction of both plasmid DNA template and purified leptospiral genomic DNA. By combining primers targeting lipL32 with the previously published primer set targeting lipL41, the sensitivity of the assay was improved to 12 copies of L. interrogans. The specificity of the LAMP assay was evaluated by using the genomic DNA from other clinically encountered bacterial species such as different strains of Orientia tsutsugamushi, Rickettsia typhi, Rickettsia conorii, Rickettsia rickettsii, Coxiella burnetii, and Bartonella bacilliformis. These genomic DNA samples were all negative in our LAMP assay. The sensitivity of the LAMP assay was very similar to that of quantitative real time PCR. Several detection methods for the amplified product of LAMP assay were performed to demonstrate the simplicity of the assay. In summary, our results have suggested that this assay is rapid, robust, and easy to perform and has the potential to be used in endemic locations.

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Rapid detection of cytochromecd1-containing nitrite reductase encoding genenirSwith loop-mediated isothermal amplification assay

10.1101/404392 ◽

2018 ◽

Author(s):

Qianqian Yang ◽

Xuzhi Zhang ◽

Xiaoyu Jiang ◽

Xiaochun Wang ◽

Yang Li ◽

...

Keyword(s):

Nitrite Reductase ◽

Genomic Dna ◽

Limit Of Detection ◽

Bacterial Species ◽

Lamp Assay ◽

Isothermal Amplification ◽

Target Sequence ◽

Bacterial Cells ◽

Conventional Pcr ◽

Loop Mediated Isothermal Amplification

AbstractThe cytochromecd1-containing nitrite reductase,nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance ofnirSis a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a new molecular biology technique termed loop-mediated isothermal amplification (LAMP) was developed to rapidly detectnirSgene using those ofPseudomonas aeruginosato optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The specificity of the assay was confirmed by the lack of amplification when using DNA from 15 other bacterial species lackingnirSgene. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, a cell-template based LAMP assay was also developed for detectingnirSgene that directly used bacterial cells as template rather than genomic DNA. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success, and bulky and sophisticated equipment were not needed. Further, thenirSgene ofP. aeruginosain spiked seawater samples could be detected with both the DNA-template based LAMP assay and the cell-template based LAMP assay, thereby demonstrating the practicality of in-field use of them. In summary, the LAMP assays described here represent a rapid, user-friendly, and cost-effective alternative to conventional PCR.

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Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison

Scientific Reports ◽

10.1038/s41598-021-91098-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Somayyeh Sedaghatjoo ◽

Monika K. Forster ◽

Ludwig Niessen ◽

Petr Karlovsky ◽

Berta Killermann ◽

...

Keyword(s):

Test Performance ◽

Genomic Dna ◽

Lamp Assay ◽

Fungal Species ◽

Isothermal Amplification ◽

Genome Comparison ◽

Performance Study ◽

Loop Mediated Isothermal Amplification ◽

Tilletia Controversa ◽

Genomic Regions

AbstractTilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.

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Loop-mediated Isothermal Amplification for Rapid Detection of Mating Types of Villosiclava virens

Plant Disease ◽

10.1094/pdis-09-21-1943-re ◽

2021 ◽

Author(s):

Xiayan Pan ◽

Xiao Wang ◽

Junjie Yu ◽

Mina Yu ◽

Huijuan Cao ◽

...

Keyword(s):

Mating Type ◽

Genomic Dna ◽

High Efficiency ◽

Lamp Assay ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification ◽

Villosiclava Virens ◽

False Smut ◽

Mating Type Genes ◽

Polymerase Chain

Rice false smut (RFS), caused by Villosiclava virens, is an important fungal disease in panicle of rice. V. virens is a heterothallic ascomycete that controlled by two opposite idiomorphs, MAT1-1 and MAT1-2. Previous study showed sexual reproduction of V. virens plays an important role in the epidemic of RFS. In this study, we have developed a loop-mediated isothermal amplification (LAMP) assay to detect mating type of V. virens easily and rapidly by using specific primers designed on the mating type genes MAT1-1-2 and MAT1-2-1, respectively. The LAMP assay required only a water/dry bath and could recognize the mating type of V. virens in just 45 min. The LAMP assay was so sensitive that could detect small amounts of V. virens genomic DNA (as low as 2.0 pg of MAT1-1, and 200.0 pg of MAT1-2), which was 10-fold more sensitive than polymerase chain reaction (PCR). In addition, the application of mating type using LAMP assay was demonstrated feasibly by assessing the genomic DNA of V. virens isolated from rice fields. The high efficiency and specificity of this LAMP assay suggested it can be used as a rapid testing tool in mating type recognition of V. virens isolates in the field.

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Rapid detection of cytochrome cd1-containing nitrite reductase encoding gene nirS of denitrifying bacteria with loop-mediated isothermal amplification assay

Scientific Reports ◽

10.1038/s41598-020-73304-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Xuzhi Zhang ◽

Qianqian Yang ◽

Qingli Zhang ◽

Xiaoyu Jiang ◽

Xiaochun Wang ◽

...

Keyword(s):

Nitrite Reductase ◽

Genomic Dna ◽

Limit Of Detection ◽

Lamp Assay ◽

Denitrifying Bacteria ◽

Isothermal Amplification ◽

Target Sequence ◽

Bacterial Cells ◽

Loop Mediated Isothermal Amplification ◽

Order Of Magnitude

Abstract The cytochrome cd1-containing nitrite reductase, nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance of nirS is a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a rapid method for detecting nirS gene with loop-mediated isothermal amplification (LAMP) was developed, using Pseudomonas aeruginosa PAO1 (P. aeruginosa PAO1) as model microorganism to optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, it was validated that P. aeruginosa PAO1 cells as well as genomic DNA could be directly used as template. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success. The nirS gene of P. aeruginosa PAO1 in spiked seawater samples could be detected with both DNA-template based LAMP assay and cell-template based LAMP assay, demonstrating the practicality of in-field use.

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Rapid identification of transgenic cotton (Gossypium hirsutum L.) plants by loop-mediated isothermal amplification

Czech Journal of Genetics and Plant Breeding ◽

10.17221/7/2011-cjgpb ◽

2011 ◽

Vol 47 (No. 4) ◽

pp. 140-148 ◽

Cited By ~ 7

Author(s):

N. Rostamkhani ◽

A. Haghnazari ◽

M. Tohidfar ◽

A. Moradi

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Lamp Assay ◽

Transgenic Cotton ◽

Rapid Identification ◽

Isothermal Amplification ◽

Detection Sensitivity ◽

Lamp Method ◽

Loop Mediated Isothermal Amplification ◽

Leaf Tissues

In an attempt to speed up the process of screening of transgenic cotton (G. hirsutum L.) plants, a visual and rapid loop-mediated isothermal amplification (LAMP) assay was adopted. Genomic DNA was extracted from fresh leaf tissues of T2 transgenic cotton containing chitinase (chi) and cry1A(b) genes. Detection of genes of interest was performed by polymerase chain reaction (PCR), LAMP and real-time PCR methods. In LAMP assay the amplification was performed after 30 min at 65°C when loop primers were involved in the reaction. The involvement of loop primers decreased the time needed for amplification. By testing serial tenfold dilutions (10–1 to 10–8) of the genes of interest, the detection sensitivity of LAMP was found to be 100-fold higher than that of PCR. The rapid DNA extraction method and LAMP assay can be performed within 30 min and the derived LAMP products can be directly observed as visually detectable based on turbidity in the reaction tube. The accuracy of LAMP method in the screening of transgenes was confirmed by PCR and real-time PCR. The developed method was efficient, rapid and sensitive in the screening of cotton transgenic plants. This method can be applied to any other crops.

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Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

Journal of Medical Microbiology ◽

10.1099/jmm.0.46071-0 ◽

2005 ◽

Vol 54 (11) ◽

pp. 1037-1041 ◽

Cited By ~ 50

Author(s):

Ryoichi Saito ◽

Yoshiki Misawa ◽

Kyoji Moriya ◽

Kazuhiko Koike ◽

Kimiko Ubukata ◽

...

Keyword(s):

Real Time ◽

Mycoplasma Pneumoniae ◽

Rapid Detection ◽

Clinical Laboratory ◽

Direct Sequencing ◽

Bacterial Species ◽

Lamp Assay ◽

Isothermal Amplification ◽

Nasopharyngeal Swab ◽

Loop Mediated Isothermal Amplification

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Mycoplasma pneumoniae was developed and evaluated. The assay specifically amplified only M. pneumoniae sequences, and no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The detection limit for this assay was found to be 2 × 102 copies, corresponding to 2–20 colour changing units of M. pneumoniae in 1 h, as observed in a real-time turbidimeter and electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis as well as direct sequencing of the amplified product. The assay was applied to 95 nasopharyngeal swab samples collected from patients or from healthy individuals, and compared to a real-time PCR assay in-house. A concordance of 100 % was observed between the two assays. The LAMP assay is easy to perform, shows a rapid reaction and is inexpensive. It may therefore be applied in the routine diagnosis of M. pneumoniae infection in the clinical laboratory.

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

Journal of Food Nutrition and Metabolism ◽

10.31487/j.jfnm.2020.02.03 ◽

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.

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Rapid Detection ofAlternariaSpecies Involved in Pear Black Spot Using Loop-Mediated Isothermal Amplification

Plant Disease ◽

10.1094/pdis-01-19-0149-re ◽

2019 ◽

Vol 103 (12) ◽

pp. 3002-3008 ◽

Cited By ~ 1

Author(s):

Xue Yang ◽

Yong-Jie Qi ◽

Mohamed N. Al-Attala ◽

Zheng-Hui Gao ◽

Xing-Kai Yi ◽

...

Keyword(s):

Genomic Dna ◽

Black Spot ◽

Lamp Assay ◽

Isothermal Amplification ◽

Cyt B ◽

Spot Disease ◽

Loop Mediated Isothermal Amplification ◽

Yellowish Green ◽

Alternaria Species ◽

Cyt B Gene

Alternaria species are the most important fungal pathogens that attack various crops as well as fruit trees such as pear and cause black spot disease. Here, a loop-mediated isothermal amplification (LAMP) assay is developed for the detection of Alternaria species. A. alternata cytochrome b (cyt-b) gene was used to design two pairs of primers and amplified a 229-bp segment of Aacyt-b gene. The results showed that LAMP assay is faster and simpler than polymerase chain reaction (PCR). LAMP assay is highly sensitive method for the detection of about 1 pg of genomic DNA of A. alternata by using optimized concentration of MgCl2(4 mM) in final LAMP reaction. In contrast, the limit of detection was 1 ng of target DNA via conventional PCR. Among the genomic DNA of 46 fungal species, only the tubes containing DNA of Alternaria spp. except A. porri, A. solani, and A. infectoria changed color from orange to yellowish green with SYBR Green I including the main pathogens of pear black spot. The yellowish green color was indicative of DNA amplification. Moreover, LAMP assay was used for testing infected tissues among 22 healthy and diseased pear tissues; the orange color changed to yellowish green for infected tissues only. Altogether, we conclude that cyt-b gene can be used for the detection of Alternaria spp. via LAMP assay, which is involved in pear black spot disease.

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