scholarly journals Time Course of Detection of Human Male DNA from Stained Blood Sample on Various Surfaces by Loop Mediated Isothermal Amplification and Polymerase Chain Reaction

2018 ◽  
Vol 2018 ◽  
pp. 1-8
Author(s):  
Panan Kanchanaphum
Keyword(s):  
Gel Electrophoresis ◽  
Time Course ◽  
Dna Amplification ◽  
Field Studies ◽  
Isothermal Amplification ◽  
Lamp Product ◽  
Loop Mediated Isothermal Amplification ◽  
Reaction Solution ◽  
Pcr Product ◽  
Pcr Products

This study explores determining the sex of humans from blood stains taken from different surfaces and compares the time course of detection with the conventional PCR, Conventional Loop Mediated Isothermal Amplification (LAMP), and LAMP-Lateral Flow Dipstick (LFD). For the DNA templates, 7 male and 7 female blood stained samples were extracted and added to LAMP and PCR reaction solution to amplify the SRY gene. The DNA samples were extracted from the following blood stained materials: cloth, wood, clay, and tile. Then, the samples were stored at room temperature for 1, 7, 30, and 60 day(s). After the DNA amplification, the gel electrophoresis process was applied to detect LAMP product. The LFD was combined with the LAMP to detect LAMP product on the male cloth samples. For the male samples, the time course of detection on the first and seventh days indicated positive for both LAMP and PCR products on all the surfaces while no DNA amplification was found on any of the female samples. On day 30, positive LAMP product was still found on all the male samples. However, it had faded on the tiles. Moreover, all the male samples, which had tested positive for PCR product, were blurred and unclear. On day 60, LAMP product was still found on all the male samples. Conversely, the PCR method resulted in no bands showing for any of the male samples. However, the LAMP-LFD method detected product on all the male samples of cloth. The results show that the LAMP is an effective, practical, and reliable molecular-biological method. Moreover, the LFD can increase the efficiency and sensitivity of the LAMP, making it more suitable for field studies because gel electrophoresis apparatus is not required.

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 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.

scholarly journals Reverse Transcription Loop-Mediated Isothermal Amplification for the Detection of Porcine Reproductive and Respiratory Syndrome Virus

2009 ◽  
Vol 21 (3) ◽  
pp. 350-354 ◽  
Author(s):  
Albert Rovira ◽  
Juan Abrahante ◽  
Michael Murtaugh ◽  
Muñoz-Zanzi Claudia
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Restriction Analysis ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Serum Samples ◽  
Loop Mediated Isothermal Amplification

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine. The objective of the current study is to investigate the feasibility of using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of PRRSV. The RT-LAMP is a recently described DNA amplification technique reported to be simple, inexpensive, fast, and accurate. The RT-LAMP reaction was set up using 2 sets of primers that were designed to detect North American and European strains of PRRSV and performed successfully in a simple heat block. The specificity of the amplified product was demonstrated by restriction analysis. The RT-LAMP was able to detect 5 different PRRSV isolates. However, the limit of detection ranged between 10 2 and 10 4 50% tissue culture infective dose/ml. The RT-LAMP was further evaluated using serum samples from animals of known infection status. The ability of RT-LAMP to detect PRRSV in serum from acutely infected animals was evaluated with 114 serum samples from 18 experimentally inoculated boars. Forty-nine of these samples tested positive by RT-LAMP, while 94 were positive by reverse transcription polymerase chain reaction (RT-PCR). The diagnostic specificity, evaluated with 100 known negative serum samples, was estimated as 99%. The feasibility of RT-LAMP to detect PRRSV was demonstrated in the current study. The RT-LAMP reaction could be performed in just 1 hr with a simple and inexpensive heat block. However, the sensitivity of this technique was significantly lower than that of RT-PCR.

scholarly journals RNase H-dependent amplification improves the accuracy of rolling circle amplification combined with loop-mediated isothermal amplification (RCA-LAMP)

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11851
Author(s):  
Takema Hasegawa ◽  
Diana Hapsari ◽  
Hitoshi Iwahashi
Keyword(s):  
Detection Limit ◽  
Rolling Circle Amplification ◽  
Dna Amplification ◽  
Rnase H ◽  
Isothermal Amplification ◽  
Rolling Circle ◽  
Loop Mediated Isothermal Amplification ◽  
Specific Amplification ◽  
Negative Condition ◽  
Rnase H2

The hybrid method upon combining rolling circle amplification and loop-mediated isothermal amplification (RCA-LAMP) was developed to quantify very small amount of different type of RNAs, such as miRNAs. RCA-LAMP can help detect short sequences through padlock probe (PLP) circularization and exhibit powerful DNA amplification. However, one of the factors that determines the detection limit of RCA-LAMP is non-specific amplification. In this study, we improved the accuracy of RCA-LAMP through applying RNase H-dependent PCR (rhPCR) technology. In this method, the non-specific amplification was suppressed by using the rh primer, which is designed through blocking the modification at the 3′end to stop DNA polymerase reaction and replacing the 6th DNA molecule from the end with RNA using RNase H2 enzyme. Traditional RCA-LAMP amplified the non-specific amplicons from linear PLP without a targeting reaction, while RCA-LAMP with rh primer and RNase H2 suppressed the non-specific amplification. Conversely, we identified the risk posed upon conducting PLP cyclization reaction using Splint R ligase in the RNA-targeting step that occurred even in the RNA-negative condition, which is another factor determining the detection limit of RCA-LAMP. Therefore, this study contributes in improving the accuracy of RNA quantification using RCA-LAMP.

scholarly journals A Stoichiometric and Pseudo Kinetic Model of Loop Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Navjot Kaur ◽  
Nikhil Thota ◽  
Bhushan Toley
Keyword(s):  
Kinetic Model ◽  
Reaction Pathway ◽  
Reaction Network ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Reaction Products ◽  
Theoretical Understanding ◽  
Loop Mediated Isothermal Amplification ◽  
Commercial Applications

<p>Loop mediated isothermal amplification (LAMP) is one of the most popular isothermal DNA amplification techniques for research and commercial applications. The LAMP mechanism is powered by strategic primer design and a strand displacement polymerase, generating products that fold over, creating loops. LAMP leads to generation of products of increasing length over time. These products containing multiple loops are conventionally called cauliflower structures. Existing literature on LAMP provides extremely limited understanding of progression of cascades of reactions involved in the reaction and it is believed that cauliflower structures of increasing length constitute a majority of the product formed in LAMP. This study presents a first of its kind stoichiometric and pseudo kinetic model to comprehend LAMP reactions in deeper depth by (i) classifying LAMP reaction products into uniquely identifiable categories, (ii) generating a condensed reaction network to depict millions of interconnected reactions occurring during LAMP, and (iii) elucidating the pathways for amplicon generation. Despite the inherent limitations of conventional stoichiometric modelling for polymerization type reactions (the network rapidly becomes too large and intractable), our model provides new theoretical understanding of the LAMP reaction pathway. The model shows that while longer length products are formed, it is the smaller length recycle amplicons that contribute more towards the exponential increase in the amount of double stranded DNA. Prediction of concentration of different types of LAMP amplicons will also contribute substantially towards informing design of probe-based assays. </p>

scholarly journals Reverse Transcription Loop-Mediated Isothermal Amplification of DNA for Detection of Potato virus Y

Plant Disease ◽  
2005 ◽  
Vol 89 (6) ◽  
pp. 605-610 ◽  
Author(s):  
Xianzhou Nie
Keyword(s):  
Potato Virus ◽  
Reverse Transcription ◽  
Potato Virus Y ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Time Saving ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

A reverse transcription loop-mediated isothermal amplification of DNA (RT-LAMP) for detection of Potato virus Y (PVY) was developed. In this procedure, a set of four primers matching a total of six sequences of the coat protein (CP) gene of PVY were designed in such a way that a loop could be formed and elongated during DNA amplification. Using PVY CP complementary DNA clones as templates, the LAMP reaction was optimized by adjusting the concentrations of MgSO4, dNTPs, and Bst DNA polymerase. The effects of fragment length of target DNA on LAMP also were investigated. Two-step and one-step RT-LAMPs were performed using RNA extracts of various PVY cultures, and the results were correlated with two-step reverse transcription polymerase chain reaction (RT-PCR) for detection of PVY. Further, the turbidity caused by precipitation of magnesium pyrophosphate formed in positive RT-LAMP reactions was used to measure the amplification by utilizing a time-saving spectrophotometric method. The one-step RT-LAMP-turbidity method gave results comparable with the two-step RT-PCR method for detection of PVY from potato leaf and tuber samples. Of the total 240 samples, 234 were diagnosed similarly by both methods.

scholarly journals Simple and rapid detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans by loop-mediated isothermal amplification assay

2018 ◽  
Vol 17 (3) ◽  
pp. 402-410 ◽  
Author(s):  
Nurul Izzati Hamzan ◽  
Fatin Hazwani Fauzi ◽  
Haslina Taib ◽  
Suharni Mohamad
Keyword(s):  
Porphyromonas Gingivalis ◽  
Rapid Detection ◽  
Detection Rate ◽  
Medical Science ◽  
Lamp Assay ◽  
Dna Amplification ◽  
Cost Effective ◽  
Aggregatibacter Actinomycetemcomitans ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification

Background: Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are two main causative agents associated with periodontitis, an inflammatory reaction of tissues around the teeth. The aim of this study was to develop and evaluate the loop-mediated isothermal amplification (LAMP) assay for simple and rapid detection of P. gingivalis and A. actinomycetemcomitans.Methods: A total of ten subgingival plaque and saliva samples were evaluated to detect the presence of both bacteria by LAMP and PCR assays. Two sets of six primers each were designed to amplify pepO and dam gene. The LAMP assay was carried out using a Loopamp DNA amplification kit in 25 μl volumes. The reaction mixture was incubated at 65oC for 60 minutes and terminated at 80oC for 5 minutes in heating block. The amplification reactions were visualized using naked eye detection and by agarose gel electrophoresis. The sensitivity of the LAMP assay was investigated ranging from 10 μg to 100fg of P. gingivalis(ATCC 33327) and A. actinomycetemcomitans (ATCC 33384).Results: The lowest detection limit of both LAMP and PCR methods were 1 ng and 10 ng of DNA, respectively. When crude template of subgingival plaques were used, P. gingivalisand A. actinomycetemcomitans were tested80% (8/10) and 60% (6/10) positive respectively through LAMP detection. Whereas by PCR, P. gingivaliswas tested 40% (4/10) positive and no significant detection rate for A. actinomycetemcomitans. When a crude template of saliva was used, P. gingivalisand A. actinomycetemcomitans were tested 70% (7/10) and 30% (3/10) positive respectively through LAMP detection. Whereas, when using PCR, there was no significant detection rate for P. gingivalisand A. actinomycetemcomitans.Conclusion: The LAMP assay using a crude template offers greater advantage as it is simple, rapid and cost-effective to detect periodontal pathogens.Bangladesh Journal of Medical Science Vol.17(3) 2018 p.402-410

scholarly journals The Effect of Polyacrilamide Gel Electrophoresis Duration on separation of Cassava SSR PCR Fragments

BioScience ◽  
2019 ◽  
Vol 3 (1) ◽  
pp. 14
Author(s):  
Nur Ayu Ramadanti ◽  
Dwi Hilda Putri
Keyword(s):  
Gel Electrophoresis ◽  
Dna Amplification ◽  
Analysis Method ◽  
Time Effects ◽  
Protein Molecules ◽  
Pcr Products ◽  
Materials Used ◽  
Field Separation ◽  
Macro Molecule ◽  
Cassava Varieties

DNA bands formed from the results of electrophoresis with Polyacrilamide gel are considered as 1 character representing 1 allele. PCR products produce multiple bands (multy bands), which indicates that there are multi alleles in the sample. Electrophoresis is a chemical analysis method based on the movement of charged protein molecules in the electric field. Separation is carried out based on differences in the size of the molecular weight and the electric charge contained by the macro-molecule. In addition, the effect of gel concentratio n, buffer and electrophoresis time also has a role in the results of electrophoresis. This study was conducted to compare the best separation time for acrilamide gel electrophoresis with the results of cassava DNA amplification. The materials used in this study are two cassava varieties, namely: Adira IV 1, Adira IV 2, Adira IV 3, Carvita 25 1, Carvita 25 2, and Carvita 25 3. Using electrophoresis by poly-acrilamide gel with two different time effects: 1 hour 30 minutes and 3 hours. The results of electrophoresis with 3 hours gave better results of DNA visualization compared to 1 hour 30 minutes.

scholarly journals Reverse Transcription Loop-Mediated Isothermal Amplification Assay with High Sensitivity to Rapid Detection of Viable Salmonella in Foods

2022 ◽  
Vol 14 (11) ◽  
Author(s):  
Dong Chen ◽  
Fangju Tian ◽  
Wanyu Liu ◽  
Jingyi Yu ◽  
Dafeng Song
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Pathogenic Bacteria ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Agarose Gel Electrophoresis ◽  
Loop Mediated Isothermal Amplification ◽  
Gene Coding ◽  
Reaction Solution ◽  
Amplification Assay

Background: Salmonella is one of the main foodborne bacterial pathogens, causing diseases and death. The study used reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect Salmonella. Objectives: To design six primers and detect Salmonella using RT-LAMP to facilitate the rapid detection of pathogenic bacteria in food. Methods: We designed six primers based on the gene coding sequences of inv A, specific to Salmonella. Each reaction solution contained 6.0 mM MgSO4, 1 M betaine, 1.6 mM dNTPs, 160 U/mL Bst DNA polymerase, 0.2 μM of both external primers, 0.8 μM of both internal primers, and 0.2 μM of both loop primers. The reaction temperature was 65°C. Results: Our amplified products were separated by 2% agarose gel electrophoresis. The detection limit was 10 CFU per reaction. Conclusions: RT-LAMP exhibited the same accuracy as the GB assay in detecting Salmonella in foods. RT-LAMP was highly specific and sensitive; hence, it may serve as an effective tool in detecting Salmonella.

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