scholarly journals Specific and rapid identification of the Pheretima aspergillum by loop-mediated isothermal amplification

2019 ◽  
Vol 39 (2) ◽  
Author(s):  
Qing Huang ◽  
Zhiwu Li ◽  
Zhiguo Ma ◽  
He Li ◽  
Runqian Mao
Keyword(s):  
Dna Polymerase ◽  
Rapid Identification ◽  
Isothermal Amplification ◽  
Coi Gene ◽  
Conventional Pcr ◽  
Lamp Product ◽  
Loop Mediated Isothermal Amplification ◽  
Animal Medicine ◽  
Site Identification ◽  
Dna Template

Abstract Guang-dilong (Pheretima aspergillum) is a traditional Chinese animal medicine that has been used for thousands of years in China. In the present study, we purposed to establish a new rapid identification method for Guang-dilong. We provided a useful technique, loop-mediated isothermal amplification (LAMP), to differentiate Guang-dilong from other species. Four specific LAMP primers were designed based on mitochondrial cytochrome c oxidase I (COI) gene sequences of Guang-dilong. LAMP reaction, containing DNA template, four primers, 10× Bst DNA polymerase reaction buffer, dNTPs, MgSO4, and Bst DNA polymerase, was completed within 60 min at 63°C. The LAMP product can be visualized by adding SYBR Green I or detected by 2% gel electrophoresis. LAMP technology was successfully established for rapid identification of Guang-dilong. In addition, DNA template concentration of 675 fg/μl was the detection limit of LAMP in Guang-dilong, which was 1000-times higher than conventional PCR. The simple, sensitive, and convenient LAMP technique is really suited for on-site identification of Guang-dilong in herbal markets.

scholarly journals Comparison of Time Course Detection of Human Male DNA from Blood Stains on Various Objects on Surface in a Natural Environment and in a Laboratory Using Loop-Mediated Isothermal Amplification (LAMP)

Scientifica ◽  
2021 ◽  
Vol 2021 ◽  
pp. 1-7
Author(s):  
Ratchanok Kumsiri ◽  
Panan Kanchanaphum
Keyword(s):  
Gel Electrophoresis ◽  
Time Course ◽  
Dna Analysis ◽  
Isothermal Amplification ◽  
Forensic Dna ◽  
Forensic Dna Analysis ◽  
Lamp Product ◽  
Loop Mediated Isothermal Amplification ◽  
Dna Template ◽  
Electrophoresis Technique

In forensic study, the biological evidence can easily degrade, especially DNA. Degraded and environmentally challenged samples can produce numerous problems in forensic DNA analysis including loss of band product. Loop-mediated isothermal amplification or LAMP is one of the DNA analysis techniques used in forensic study. This study explores the limitations of the efficiency of the LAMP technique on abandoned DNA. For the DNA template, 8 male and 2 female blood-stained samples were taken from the surfaces, namely, brick, cloth, and tile from inside, and buried outside the laboratory. The LAMP reaction was used to amplify the SRY gene for detecting male DNA. All the blood-stained samples were stored for 1, 7, 15, 30, and 45 day (s). The LAMP product from the blood-stained samples on all the surfaces that were kept in a laboratory was detected using the gel electrophoresis technique from day 1 until day 45. However, the LAMP product on day 30 and 45 was smear and dim. The LAMP product from the blood-stained samples buried outside the laboratory was observed using the gel electrophoresis technique within day 30 (smear and dim). To increase the efficiency of detection, the qLAMP technique detected product on all the male samples from all the surfaces buried outside the laboratory for 45 days. The results indicate that this LAMP condition was possible detecting male DNA and the environmental factors are the main influence on the sensitivity of the LAMP technique. In addition, the qLAMP technique can increase the capacity and sensitivity of the detection.

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 Rapid identification of Aconitum plants based on loop-mediated isothermal amplification assay

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Masashi Kitamura ◽  
Akira Kazato ◽  
Tadashi Yamamuro ◽  
Hirokazu Ando ◽  
Yohei Sasaki ◽  
...  
Keyword(s):  
Rapid Identification ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay

scholarly journals Monitoring Methyl Benzimidazole Carbamate-Resistant Isolates of the Cucurbit Powdery Mildew Pathogen, Podosphaera xanthii, Using Loop-Mediated Isothermal Amplification

Plant Disease ◽  
2019 ◽  
Vol 103 (7) ◽  
pp. 1515-1524 ◽  
Author(s):  
Alejandra Vielba-Fernández ◽  
Antonio de Vicente ◽  
Alejandro Pérez-García ◽  
Dolores Fernández-Ortuño
Keyword(s):  
Amino Acid ◽  
Powdery Mildew ◽  
Amino Acid Change ◽  
Color Change ◽  
Isothermal Amplification ◽  
Podosphaera Xanthii ◽  
Lamp Product ◽  
Loop Mediated Isothermal Amplification ◽  
Cucurbit Powdery Mildew ◽  
Β Tubulin

Powdery mildew, caused by the fungus Podosphaera xanthii, is one of the most economically important diseases affecting cucurbit crops in Spain. Currently, chemical control offers the most efficient management of the disease; however, P. xanthii isolates resistant to multiple classes of site-specific fungicides have been reported in the Spanish cucurbit powdery mildew population. In previous studies, resistance to the fungicides known as methyl benzimidazole carbamates (MBCs) was found to be caused by the amino acid substitution E198A on β-tubulin. To detect MBC-resistant isolates in a faster, more efficient, and more specific way than the traditional methods used to date, a loop-mediated isothermal amplification (LAMP) system was developed. In this study, three sets of LAMP primers were designed. One set was designed for the detection of the wild-type allele and two sets were designed for the E198A amino acid change. Positive results were only obtained with both mutant sets; however, LAMP reaction conditions were only optimized with primer set 2, which was selected for optimal detection of the E198A amino acid change in P. xanthii-resistant isolates, along with the optimal temperature and duration parameters of 65°C for 75 min, respectively. The hydroxynaphthol blue (HNB) metal indicator was used for quick visualization of results through the color change from violet to sky blue when the amplification was positive. HNB was added before the amplification to avoid opening the lids, thus decreasing the probability of contamination. To confirm that the amplified product corresponded to the β-tubulin gene, the LAMP product was digested with the enzyme LweI and sequenced. Our results show that the LAMP technique is a specific and reproducible method that could be used for monitoring MBC resistance of P. xanthii directly in the field.

Rapid identification of RNA‐interference‐based resistance to Bean golden mosaic virus in transgenic common beans via loop‐mediated isothermal amplification

Crop Science ◽  
2020 ◽  
Vol 60 (6) ◽  
pp. 3004-3012
Author(s):  
Nara Cristina Teixeira ◽  
Adriane Wendland ◽  
Maythsulene Inácio de Souza Oliveira ◽  
Livia Teixeira Duarte Brandão ◽  
Thiago Lívio Pessoa Oliveira Souza ◽  
...  
Keyword(s):  
Rna Interference ◽  
Mosaic Virus ◽  
Rapid Identification ◽  
Isothermal Amplification ◽  
Common Beans ◽  
Bean Golden Mosaic Virus ◽  
Loop Mediated Isothermal Amplification

scholarly journals Quantitative LAMP and PCR Detection of Salmonella in Chicken Samples Collected from Local Markets around Pathum Thani Province, Thailand

2020 ◽  
Vol 2020 ◽  
pp. 1-6 ◽  
Author(s):  
Virun Vichaibun ◽  
Panan Kanchanaphum
Keyword(s):  
Efficient Method ◽  
Food Chain ◽  
Dna Detection ◽  
Pcr Detection ◽  
Isothermal Amplification ◽  
Artificial Infection ◽  
Conventional Pcr ◽  
Loop Mediated Isothermal Amplification ◽  
Contaminated Food ◽  
Pcr Method

Salmonella is a bacterium that infects people when they consume contaminated food or liquids. To prevent humans from becoming ill, it is useful to have an efficient method of detecting Salmonella before the disease is passed on through the food chain. In this research, the efficiency of Salmonella detection was compared using the following four methods: conventional loop-mediated isothermal amplification (LAMP), PCR, quantitative LAMP (qLAMP), and qPCR. The artificial infection of chicken samples started with incubating of 10 mL of 108 CFU of S. typhimurium for 6 hr. and enriching for 2 hr. to represent real contamination of the samples. The results show that the sensitivity of Salmonella DNA detection in PCR, qPCR, LAMP, and qLAMP were 50 ng, 5 ng, 50 pg, and and 500 fg, respectively. Thirty samples of 10 g chicken were collected from 10 markets in Pathum Thani, Thailand; then, the infection was detected. The conventional LAMP, qLAMP, and qPCR methods detected Salmonella in all the chicken samples. However, the conventional PCR method detected Salmonella infection in only eight of the samples. Overall, the qLAMP method had the highest sensitivity of Salmonella DNA detection.

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