NAIL: Nucleic Acid detection using Isotachophoresis and Loop-mediated isothermal amplification.

Specific, sensitive and rapid Curcuma longa turmeric powder authentication in commercial food using loop-mediated isothermal nucleic acid amplification.

With the increase of patients suffering from immune deficiency infections also increased pulmonary fungi even in people with defective immune system can cause fatal and lethal candidiasis. The timely diagnosis of pulmonary candidiasis is one of the problems that has been detected. Polymerase chain reaction (PCR) test and Loop mediated isothermal amplification (LAMP) method optimized on the basis of α INT1 gene and then sensitivity and specificity were evaluated. LAMP is a novel nucleic acid amplification technique with high specificity and sensitivity which has been done under isothermal condition. Samples were the bronchoalveolar lavage suspected of tuberculosis (TB) reviews for TB disease negative have been reported. DNA extraction carried out by standard phenol/chloroform method on samples and PCR test and LAMP was done. PCR and LAMP testing was performed on samples and products of 441 bp were amplified and observed with agarose gel electrophoresis. At the end of the LAMP reaction, SYBR Green was used for identifying negative and positive results. Among the 60 quantities sera, only 7 cases were PCR positive but 8 cases were LAMP positive. In comparison, between LAMP and PCR, the LAMP technique in spite of its simplicity, high sensitivity and specificity, could be an appropriate replacement for PCR.

Dual-target CRISPR-Cas12 diagnostics based on asymmetrically chemical-modified DNA probe.

Isothermal nucleic acid amplification techniques: A comprehensive overview on their principle & applications as alternative to PCR

Efficacy of loop mediated isothermal amplification (LAMP) assay for the laboratory identification of Mycobacterium tuberculosis isolates in a resource limited setting

Sequence-specific isothermal nucleic acid amplification techniques are ideally suited for use in molecular diagnostic applications because they do not require thermal cycling equipment and the reactions are typically fast. One of the most widely cited isothermal techniques is termed loop-mediated isothermal amplification (LAMP). This protocol allows amplification times as fast as 5 to 10 min. Furthermore, various methodologies to detect amplification have been applied to LAMP to increase its utility for the point-of-care market. Basic LAMP protocols are provided herein for detection of specific DNA and RNA targets, along with a method to perform multiplex LAMP reactions, permitting even greater flexibility from this powerful technique.

Highly efficient loop cleavage for human papillomavirus detection with a novel thermophilic Argonaute from Thermus brockianus.

BackgroundMalaria is a globally distributed infectious disease. According to the World Health Organization, Angola is one of the six countries that account for over half the global malaria burden in terms of both malaria cases and deaths. Diagnosis of malaria still depends on microscopic examination of thin and thick blood smears and rapid diagnostic tests (RDTs), which often lack analytical and clinical sensitivity. Molecular methods could overcome these disadvantages. The aim of this study was to evaluate, for the first time to our knowledge, the performance of a loop-mediated isothermal amplification (LAMP) for the diagnosis of malaria in an endemic area in Cubal, Angola, and to assess the reproducibility at a reference laboratory.MethodsA total of 200 blood samples from patients attended at Hospital Nossa Senhora da Paz, Cubal, Angola, were analysed for Plasmodium spp. detection by microscopy, RDTs, and LAMP. LAMP assay was easily performed in a portable heating block, and the results were visualized by a simple colour change. Subsequently, the samples were sent to a reference laboratory in Spain to be reanalysed by the same colorimetric LAMP assay and also in real-time LAMP format.ResultsIn field tests, a total of 67/200 (33.5%) blood samples were microscopy-positive for Plasmodium spp., 98/200 RDT positive, and 112/200 (56%) LAMP positive. Using microscopy as reference standard, field LAMP detected more microscopy-positive samples than RDTs (66/67; 98% vs. 62/67; 92.5%). When samples were reanalysed at a reference laboratory in Spain using both colorimetric and real-time assays, the overall reproducibility achieved 84.5%.ConclusionsThis is the first study to our knowledge in which LAMP has been clinically evaluated on blood samples in a resource-poor malaria-endemic area. The colorimetric LAMP proved to be more sensitive than microscopy and RDTs for malaria diagnosis in field conditions. Furthermore, LAMP showed an acceptable level of reproducibility in a reference laboratory. The possibility to use LAMP in a real-time format in a portable device reinforces the reliability of the assay for molecular diagnosis of malaria in resource-poor laboratories in endemic areas.Graphical

Objective To develop a method for detecting the DNA of Giardia lamblia by loop-mediated isothermal amplification (LAMP). Methods The DNA was extracted from cultured in vitro G. lamblia trophozoites. According to the DNA sequence of G. lamblia in GenBank and the principal of LAMP, 4 specific primers were designed for LAMP assay detection on G. lamblia, the DNA of Cryptosporidium andersoni and Plasmodium falciparum were also detected as control, and pure water without DNA as negative control. The LAMP products were stained by SYBR green I and analyzed by agarose gel electrophoresis. Results After LAMP reaction, the positive signal from G. lamblia DNA turned green while the other two kinds DNA of pathogens and negative control kept brown. Moreover,electrophoresis analysis showed the characterized ladder of the bands for LAMP product of G. lamblia. By contrast,no bands observed for the LAMP products of andersoni, Plasmodium fakiparum and water. Conclusion A simple method for detecting G. lamblia by LAMP assay was established. Key words: Loop-mediated isothermal amplification; Giardia lamblia; Cryptosporidium andersoni

African swine fever (ASF) is a contagious viral disease, which can cause up to 100% mortality among domestic pigs. Pig production is growing rapidly in Uganda among East African countries and is not only a source of food but also an important income for many people living in the rural areas. Field diagnosis of ASF depends only on clinical signs and has to be confirmed in the laboratory since the clinical signs are not pathognomonic. Diagnostic techniques for ASF are focused on serological tests for detection of antigen and antibody, genomic DNA detection by polymerase chain reaction (PCR), and on virus isolation and localization in clinical samples. There have been many recent reports of ASF outbreaks in Uganda yet laboratory diagnosis is limited due to the high cost and expertise required. This work reports the evaluation and application of a loop-mediated isothermal amplification (LAMP) test for detecting African swine fever virus (ASFV) DNA based on the topoisomerase II gene. Thirty (30) tissue samples obtained from suspected ASF outbreaks were collected from different regions of Uganda. The tissue samples were found to have lesions consistent with ASF. One hundred and eighty eight (188) additional blood samples were obtained from the abattoir and field surveillance. Six primers targeting the topoisomerase II gene were used. The sensitivity and specificity of LAMP and OIE recommended diagnostic PCR were compared. The LAMP assay is rapid with results obtained within 1 h (45-60 min). The sensitivity of LAMP for the detection of ASFV was 100% (95% CI: 91.78-100) while the specificity was 44% (95% CI: 36.52-51.69). The Kappa statistic for level of agreement between PCR and LAMP test in the detection of ASFV was 23.7% (95% CI: 16.42-30.91). This Kappa value indicated a fair agreement between the two assays. No cross reaction was observed with Porcine circovirus type 2 virus and E. coli isolated from pigs in Uganda. This is the first study evaluating and applying the LAMP assay in the detection of ASF in domestic pigs in Uganda. The LAMP assay was found to be more sensitive than PCR. Due to its simplicity, sensitivity and specificity, the LAMP assay has the potential for use in the diagnosis and routine surveillance of ASF in Uganda.   Key words: African swine fever virus, loop-mediated isothermal amplification (LAMP), polymerase chain reaction (PCR), sensitivity, specificity, topoisomerase II gene.

Prompt and accurate diagnosis of gastrointestinal tuberculosis (GITB) is highly challenging. Current conventional techniques lack sensitivity and are time-consuming. Multi targeted loop-mediated isothermal amplification (LAMP) using two targets (IS6110 and MPB64) is a promising technique for rapid diagnosis of TB. LAMP test using IS6110 and MPB64 targets for M. tuberculosis complex was performed on ileocecal biopsy samples of 35 clinically suspected patients of GITB and 30 ileocecal biopsy samples of non-tuberculosis control subjects. Results of IS6110 LAMP and MPB64 LAMP were compared with IS6110 PCR and culture INFERENCE: Overall LAMP test (using any of the two targets) had a sensitivity and specificity of 100 and 100%, respectively, for confirmed GITB (five culture positive) cases and 85.71 and 100%, respectively, for clinically suspected 30 ileocecal biopsy samples of GITB. Sensitivity of IS6110 LAMP, MPB64 LAMP and IS6110 PCR for clinically suspected cases was 22 (73.33%), 24 (80%) and 21 (70%), respectively. In total 35 GITB patients, the overall sensitivity of microscopy, culture, IS6110 PCR, IS6110 LAMP, MPB64 LAMP and the multi-targeted LAMP assay (if any of the two targets were used) were 5.71, 14.28, 68.57, 74.28, 82.85 and 85.71%, respectively. Specificity of all the tests was 100%. There were three cases which were missed by IS6110 LAMP and two cases by MPB64 LAMP. Multi-targeted LAMP is a promising technique for rapid and accurate diagnosis of GITB.

Edwarsiela ictaluri is a potential bacteria as a pathogen causing Enteric Septicemia of Catfish (ESC) and also still a big problem in catfish because it can cause death to 100%. This study aims to develop diagnosis DNA based on molecular using Loop Mediated Isothermal Amplification. Method (LAMP). Detection DNA of E. ictaluri bacteria from 9 isolates was extracted at Fish Health Laboratory, in Depok. used four pieces of specific primers from Eip 18 gene of E. ictaluri. DNA amplification, for LAMP reactions without a thermocycler machine, using only an incubator/water bath/oven, at 65°C for 1 hour. The result of detection with LAMP, showed that 8 isolates were positive of E. ictaluri marked by SYBR green I color change from orange to green. While 1 isolate was negatively marked with SYBR Green remain orange. The LAMP results are also verified with PCR using outer primer F3 and B3 which showed positive E. ictaluri. LAMP can detect ESC disease simple, quickly, easily, economically in 1 hour compared by PCR and LAMP method can be used as an alternative diagnostic test for fish diseases, especially in areas with limited laboratory infrastructure.

Background/aim Tuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure.Materials and methods Sputum samples were homogenized with 5% trypsin solution in CaCl2 to obtain DNA. DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB’s Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w/v agarose gel electrophoresis methods were used.ResultsOptimum amplification temperature for LAMP was found to be 71.4 °C. The detection limit of the method was 102 CFU/mL and sensitivity was determined 100% compared to five different Mycobacterium species. Conclusion The current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field.

Conventional diagnosis of filarial infections is based on morphological identification of microfilariae using light microscopy and requires considerable expertise, is time-consuming, and can be subjective. Loop-mediated isothermal amplification (LAMP) has advantages over microscopy or PCR because of its operational simplicity, rapidity and versatility of readout options. LAMP assays represent a major step forward in improved filarial diagnostic tools suitable for low resource settings and field applicability. The study goal was to retrospectively evaluate the performance and suitability of the O-150, RF4, and Mp419 LAMP assays for diagnosing Onchocerca volvulus, Loa loa and Mansonella perstans infections, respectively, in humans and vectors under experimental and natural field conditions. Surveys were conducted in four health districts of Cameroon using skin snip and thick blood film methods to detect skin (O. volvulus) and blood (L. loa and M. perstans) dwelling microfilaria in humans. Engorged vectors (Simulium spp., Chrysops spp., and Culicoides spp.) were evaluated by LAMP. Dissected, wild-caught vectors were also analyzed. LAMP showed a prevalence of 40.4% (O. volvulus), 17.8% (L. loa) and 36.6% (M. perstans) versus 20.6% (O. volvulus), 17.4% (L. loa) and 33.8% (M. perstans) with microscopy. Simulium spp. were dissected for microscopy and pooled for LAMP. The O-150 LAMP assay infection rate was 4.3% versus 4.1% by microscopy. Chrysops spp. were dissected and analyzed individually in the LAMP assay. The RF4 LAMP assay infection rate was 23.5% versus 3.3% with microscopy. The RF4 LAMP assay also detected parasites in Chrysops spp. fed on low microfilaremic volunteers. The Mp419 LAMP assay infection rate was 0.2% for C. milnei and 0.04% for C. grahamii, while three other species were LAMP-negative. The sensitivity, species specificity, rapidity and ease of its use of these filarial LAMP assays, and validation of their performance in the field support use as alternatives to microscopy as diagnostic and surveillance tools in global health programs aimed to eliminate onchocerciasis.

Development of a novel genotype-specific loop-mediated isothermal amplification technique for Hepatitis B virus genotypes B and C genotyping and quantification