scholarly journals Development of Loop-Mediated Isothermal Amplification for Detection ofLeifsonia xylisubsp.xyliin Sugarcane

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Jing Liu ◽  
Liping Xu ◽  
Jinlong Guo ◽  
Rukai Chen ◽  
Michael Paul Grisham ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Conventional Pcr ◽  
Time Saving ◽  
Visual Judgment ◽  
Coryneform Bacterium ◽  
Loop Mediated Isothermal Amplification ◽  
Total Dna ◽  
Sensitive Method

Ratoon stunt, caused by the xylem-limited coryneform bacteriumLeifsonia xylisubsp.xyli(Lxx), is a deep bacteriosis and prevalent in most of sugarcane-producing countries. Based on loop-mediated isothermal amplification (LAMP), we developed a method for detectingLxx. The major advantages of the LAMP method are visual judgment by color and time saving with only 60 min for identification ofLxxand without the need for costly PCR apparatus and gel scanner. In the present study, positive and negative samples detected by the LAMP method were clearly distinguishable. When total DNA extracted from internode juice was used as the template, the sensitivity of LAMP was 10 times higher than that of the conventional PCR detection. The LAMP assay is a highly specific, rapid, and sensitive method for the diagnosis of ratoon stunt caused byLxxin sugarcane. This is the first report of LAMP-based assay for the detection ofLxxin sugarcane.

Development and Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Method for Detecting Foodborn Salmonella in Raw Milk

2013 ◽  
Vol 647 ◽  
pp. 577-582 ◽  
Author(s):  
Yong Zhen Wang ◽  
De Guo Wang
Keyword(s):  
Reference Method ◽  
Lamp Assay ◽  
Raw Milk ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Conventional Pcr ◽  
Detection Level ◽  
Loop Mediated Isothermal Amplification ◽  
Milk Samples ◽  
Food Borne

In present study, we reported the performance of a Loop-mediated isothermal amplification (LAMP) assay detecting food-borne pathogen Salmonella. Three pairs of primers were specially designed for recognizing eight distinct sequences on the target invA gene. Time and temperature conditions for amplification of Salmonella were optimized to be 40 min at 61°C. The LAMP assay gave with artificially contaminated raw milk samples detection limit level of 142 CFU/ml which corresponds to 6-9 cells per reaction tube, while the detection level of conventional PCR was 103 CFU/ml. Data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 89.58% concordance with the ISO 6579 reference method for the samples without enrichment and 100% concordance for the samples after enrichment.

A rapid, sensitive, specific and visual detection of Salmonella in retail meats using the loop-mediated isothermal amplification (LAMP), targeting the invA gene

2021 ◽  
Author(s):  
Can Wang ◽  
Ziheng Xu ◽  
Xuejiao Hou ◽  
Min Wang ◽  
Chenyu Zhou ◽  
...  
Keyword(s):  
Food Safety ◽  
Visual Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
National Standard ◽  
Lamp Method ◽  
Analytical Sensitivity ◽  
Conventional Pcr ◽  
Inva Gene ◽  
Loop Mediated Isothermal Amplification

Salmonella is one of the major pathogenic bacteria causing food-borne diseases. The rapid detection of Salmonella in food is of great significance to food safety. In this study, the loop-mediated isothermal amplification (LAMP) method was developed and the primers were designed targeting the invA gene of Salmonella. Then, the standard samples of recombinant invA-plasmid and 100 retail meat samples were tested by LAMP and compared with the results tested by the conventional PCR and the routine China National Food Safety Standard Methods for Food Microbiological Examination-Salmonella (GB/T4789.4-2016), respectively. The results showed that Salmonella strains of 8 different serotypes were amplified successfully by the developed LAMP assay. And, it was 1000-fold more sensitive than the conventional PCR with the analytical sensitivity of 8×102 copies/μL of the standard sample of invA-plasmid. The results were visualized directly by adding Calcein/MnCl2 into the LAMP reaction tube and the positively amplified products turned green after an incubation of 2 min. In the parallel detection, the positive rate of Salmonella by the LAMP assay was highly correlated with the routine China national standard method. The results of the study demonstrated that the developed LAMP assay is a simple, rapid, strongly specific, highly sensitive and visual detection method for Salmonella.

scholarly journals Rapid detection of Pecan Root-Knot Nematode, Meloidogyne partityla, in laboratory and field conditions using loop-mediated isothermal amplification

2020 ◽  
Author(s):  
Sumyya Waliullah ◽  
Jessica Bell ◽  
Tammy Stackhouse ◽  
Ganpati Jagdale ◽  
Abolfazl Hajihassani ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Nematode Species ◽  
Conventional Polymerase Chain Reaction ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Field Conditions ◽  
Lamp Method ◽  
Conventional Pcr ◽  
Root Knot Nematode ◽  
Loop Mediated Isothermal Amplification

AbstractMeloidogyne partityla is the dominant root-knot nematode (RKN) species parasitizing pecan in Georgia. This species is known to cause a reduction in root growth and a decline in yields from mature pecan trees. Rapid and accurate diagnosis of this RKN is required to control this nematode disease and reduce losses in pecan production. In this study, a loop-mediated isothermal amplification (LAMP) method was developed for simple, rapid and on-site detection of M. partityla in infested plant roots and validated to detect the nematode in laboratory and field conditions. Specific primers were designed based on the sequence distinction of internal transcribed spacer (ITS)-18S/5.8S ribosomal RNA gene between M. partityla and other Meloidogyne spp. The LAMP detection technique could detect the presence of M. partityla genomic DNA at a concentration as low as 1 pg, and no cross reactivity was found with DNA from other major RKN species such as M. javanica, M. incognita and M. arenaria, and M. hapla. We also conducted a traditional morphology-based diagnostic assay and conventional polymerase chain reaction (PCR) assay to determine which of these techniques was less time consuming, more sensitive, and convenient to use in the field. The LAMP assay provided more rapid results, amplifying the target nematode species in less than 60 min at 65°C, with results 100 times more sensitive than conventional PCR (~2-3 hrs). Morphology-based, traditional diagnosis was highly time-consuming (2 days) and more laborious than conventional PCR and LAMP assays. These features greatly simplified the operating procedure and made the assay a powerful tool for rapid, on-site detection of pecan RKN, M. partityla. The LAMP assay will facilitate accurate pecan nematode diagnosis in the field and contribute to the management of the pathogen.

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

2011 ◽  
Vol 47 (No. 4) ◽  
pp. 140-148 ◽  
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 T<sub>2</sub> 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&deg;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<sup>&ndash;1</sup> to 10<sup>&ndash;8</sup>) 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.

scholarly journals Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients

2008 ◽  
Vol 57 (4) ◽  
pp. 439-443 ◽  
Author(s):  
Basu Dev Pandey ◽  
Ajay Poudel ◽  
Tomoko Yoda ◽  
Aki Tamaru ◽  
Naozumi Oda ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Lamp Method ◽  
Rrna Gene ◽  
Predictive Values ◽  
Loop Mediated Isothermal Amplification ◽  
System A

A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5–97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 %, respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.

Rapid Detection of Mycoplasma pneumoniae by Loop-Mediated Isothermal Amplification (LAMP) in Clinical Respiratory Specimens

2019 ◽  
Author(s):  
Maryam ARFAATABAR ◽  
Narjes NOORI GOODARZI ◽  
Davoud AFSHAR ◽  
Hamed MEMARIANI ◽  
Ghasem AZIMI ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Culture Method ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Culture Methods ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Respiratory Specimens

  Background: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP) worldwide, especially among children and debilitated populations. The present study aimed to investigate a loop-mediated isothermal amplification (LAMP) technique for rapid detection of M. pneumoniae in clini-cal specimens collected from patients with pneumonia. Methods: Throat swabs were collected from 110 outpatients who suffered from pneumonia. Throat swab samples were obtained from patients referred to the hospital outpatient clinics of Tehran University hospitals, Iran in 2017. The presence of M. pneumoniae in the clinical specimens was evaluated by LAMP, PCR and culture methods. Sensitivity and specificity of the LAMP and PCR assays were also determined. Results: Out of 110 specimens, LAMP assay detected M. pneumoniae in 35 specimens. Detection limit of the LAMP assay was determined to be 33fg /μL or ~ 40 genome copies/reaction. Moreover, no cross-reaction with genomic DNA from other bacteria was observed. Only 25 specimens were positive by the culture method. The congruence between LAMP assay and culture method was ‘substantial’ (κ=0.77). Specificity and sensitivity of LAMP assay were 88.2%, 100% in compare with culture. However, the con-gruence between LAMP assay and PCR assay was ‘almost perfect’ (κ=0.86). Specificity and sensitivity of LAMP assay were 92.5%, 100% in compare with PCR. Conclusion: Overall, the LAMP assay is a rapid and cost-efficient laboratory test in comparison to other methods including PCR and culture. Therefore, the LAMP method can be applied in identification of M. pneumoniae isolates in respiratory specimens.

scholarly journals Detection of Haemophilus parasuis isolates from South China by loop-mediated isothermal amplification and isolate characterisation

2012 ◽  
Vol 79 (1) ◽  
Author(s):  
Jian-min Zhang ◽  
Hai-yan Shen ◽  
Ming Liao ◽  
Tao Ren ◽  
Li-li Guo ◽  
...  
Keyword(s):  
16S Rrna ◽  
South China ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Economic Losses ◽  
Lamp Method ◽  
Haemophilus Parasuis ◽  
Loop Mediated Isothermal Amplification ◽  
Glässer’S Disease ◽  
Glasser's Disease

Haemophilus parasuis is the etiological agent of Glässer’s disease, which is characterised by fibrinous polyserositis, meningitis and polyarthritis, causing severe economic losses to the swine industry. In this study, a loop-mediated isothermal amplification (LAMP) test was developed to improve the specificity, facility and speed of diagnosis of H. parasuis isolates. The LAMP assay rapidly amplified the target gene within 50 min incubation at 63 °C in a laboratory water bath. The LAMP amplicon could be visualised directly in the reaction tubes following the addition of SYBR Green I dye. The detection limit of this LAMP method was 10 CFU/mL, which was 10 times more sensitive than the earlier 16S rRNA polymerase chain reaction (PCR) test conducted by Oliveira, Galina and Pijoan (2001), and no cross-reactivity was observed from other non-H. parasuis strains. This LAMP test was evaluated further on 187 clinical specimens from pigs suspected of being infected with H. parasuis. Forty-three were found positive by bacterial isolation of H. parasuis, as well as by the 16S rRNA PCR and LAMP tests. The 43 H. parasuis isolates were classified into 9 serovars and had 37 genetic patterns when analysed by pulsed-field gel electrophoresis (PFGE). This displayed that various H. parasuis serovars and genotypes were widely distributed in South China. Therefore, the speed, specificity and sensitivity of the LAMP test, the lack of a need for expensive equipment, and the visual readout showed great potential for a correct clinical diagnosis of H. parasuis in favour of controlling Glässer’s disease.

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

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.

scholarly journals Loop-Mediated Isothermal Amplification (LAMP) Assay to De-tect Toxoplasmosis in Schizophrenia Patients

2020 ◽  
Author(s):  
Hadi MIRAHMADI ◽  
Raheleh HASANZADEH ◽  
Hamid MALEK RAEESI ◽  
Shirzad FALLAHI ◽  
Mahdi KHOSHSIMA SHAHRAKI ◽  
...  
Keyword(s):  
Nested Pcr ◽  
High Efficiency ◽  
Parasitic Infection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Intermediate Hosts ◽  
Cross Sectional ◽  
Loop Mediated Isothermal Amplification ◽  
Schizophrenic Patients

Background: Toxoplasma gondii (T. gondii) causes an important parasitic infection known as toxoplasmosis, which is a globally distributed important zoonosis. One of the major serious characteristics of T. gondii is its ability to manipulate the behavior of intermediate hosts. We performed a cross-sectional study to determine toxoplasmosis in schizophrenic patients, as one of the major neuropsychiatric disorders, using loop-mediated isothermal amplification (LAMP) technic by targeting parasite B1 gene. Methods: Blood samples were taken from 118 schizophrenic patients hospitalized in tow hospitals including Baharan, Clinic of Psychiatric Ali-ibn-Abi-Talib Hospital (in Zahedan City), and Amir-al Momenin Psychiatric Hospital (in Zabol City), Sistan and Baluchestan Province, southeast Iran in 2016. They were analyzed using LAMP, and compared with the previous data of nested-PCR and serology. Results: Out of the 118 schizophrenic individuals, 56 patients (47.4%) were found to be infected with T. gondii. The diagnosis of toxoplasmosis was confirmed in 41 patients (34.7%) via the nested-PCR. The seroprevalence of toxoplasmosis in schizophrenic patients was 55.9% (66/118). Conclusion: We found a high efficiency of LAMP method in identifying toxoplasmosis and its high prevalence among schizophrenic patients. Our findings could provide viable offer implications for the prevention of schizophrenia.

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