Loop-Mediated Isothermal Amplification Combined with Lateral Flow Dipstick for On-Site Diagnosis of African Swine Fever Virus.
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ABSTRACT:
SUBMITTER: Zuo L
PROVIDER: S-EPMC8087745 | biostudies-literature | 2021 Apr
REPOSITORIES: biostudies-literature
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Loop-Mediated Isothermal Amplification Combined with Lateral Flow Dipstick for On-Site Diagnosis of African Swine Fever Virus.
Zuo Lei L Song Zengxu Z Zhang Yi Y Zhai Xiwen X Zhai Yaru Y Mei Xueran X Yang Xin X Wang Hongning H
Virologica Sinica 20201106 2
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Project description:IntroductionTo eradicate Helicobacter pylori (H. pylori) and reduce the risk of gastric cancer, a sensitive, specific, convenient, and simple detection method is needed. This study aimed to establish a novel loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for H. pylori detection.MethodsLAMP primer design software was used to design primers for the conserved sites of the H. pylori ureB gene. UreB-FIP-labeled biotin was used for LAMP amplification, and FAM-labeled probes were specifically hybridized with LAMP amplification products, which were then detected by LFD. In addition, a clinical study was conducted to assess LAMP-LFD in 20 fecal samples.ResultsThe results of the optimization indicated that H. pylori could be specifically detected by LFD without cross-reaction with other non-H. pylori bacteria when the LAMP was performed at 65°C for 60 min. The lower limit of the detection method was 102 copies/μL, which was 100 times the sensitivity of polymerase chain reaction (PCR). H. pylori-positive fecal samples were detected by LAMP-LFD in 13/20 patients.DiscussionIn conclusion, a new LAMP-LFD assay has been fully established and confirmed for H. pylori detection. The entire process can be completed in approximately 1.5 h, with the advantages of strong specificity, high sensitivity, and simple operation. This study provides a novel potential method for the detection of H. pylori in the clinical settings of primary hospitals and low-resource countries.
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Project description:Tuberculosis (TB) is a communicable disease caused by the bacterium Mycobacterium tuberculosis (MTB) and is a persistent problem in the developing countries. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here, a LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect IS6110 gene of M. tuberculosis specifically and rapidly. The reaction was optimized at 63°C for 60?min, and the amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5?min was detected at the LFD test line 5?min after application. Excluding the step of DNA extraction, the test results could be generated approximately within 1?h. In addition to the advantage of short assay time, this technique could avoid the contact of carcinogenic ethidium bromide due to the exclusion of the electrophoresis analysis step. Furthermore, the data indicated that LAMP-LFD could detect M. tuberculosis genomic DNA as little as 5?pg. The technique showed a significant specificity since no cross-hybridization to M. intracellulare (MIC), M. fortuitum (MFT), M. avium (MAV), M. kansasii (MKS), and M. gordonae (MGD) genomic DNAs was observed. In the clinical unknown samples test, the sensitivity of LAMP-LFD was 98.92 ? % and the specificity was 100 ? % compared to those of the standard culture assay. Based on its sensitivity, specificity, rapidity, low cost, and convenience, LAMP-LFD could be applicable for use in both laboratories and epidemiological surveys of MTB.
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Project description:BACKGROUND: Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions. RESULTS: A recent DNA amplification technique known as Loop Mediated Isothermal Amplification (LAMP) was adapted for the detection of Candidatus Liberibacter asiaticus. This methodology was combined with a Lateral Flow Dipstick (LFD) device for visual detection of the resulting amplicons, eliminating the need for gel electrophoresis. The assay was highly specific for the targeted bacterium. No cross-reaction was observed with DNA from any of the other phytopathogenic bacteria or fungi assayed. By serially diluting purified DNA from an infected plant, the sensitivity of the assay was found to be 10 picograms. This sensitivity level was proven to be similar to the values obtained running a real time PCR in parallel. This methodology was able to detect Candidatus Liberibacter asiaticus from different kinds of samples including infected citrus plants and psyllids. CONCLUSIONS: Our results indicate that the methodology here reported constitutes a step forward in the development of new tools for the management, control and eradication of this destructive citrus disease. This system constitutes a potentially field-capable approach for the detection of the most relevant HLB-associated bacteria in plant material and psyllid vectors.
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Project description:BackgroundClostridium piliforme (causative agent of Tyzzer disease) infects various animals, including primates, and hence a threat to animal and human health worldwide. At present, it is detected using traditional methods, such as path morphology, polymerase chain reaction and enzyme-linked immunosorbent assay. Therefore, it is necessary to develop convenient, efficient visual molecular biological methods for detecting C. piliforme.ObjectivesTo establish a method with good specificity, high sensitivity and simple operation for the detection of C. piliforme.MethodsIn this study, we designed internal and external primers based on the conserved 23S rRNA region of C. piliforme to develop a biotin-labelled diarrhoea-suffered loop-mediated isothermal amplification (LAMP) system for detecting of C. piliforme and assessed the specificity, sensitivity and repeatability of the LAMP system.ResultsThe LAMP system did not exhibit cross-reactivity with 24 other common pathogenic species, indicating that it had good specificity. The minimum concentration of sensitivity was 1 × 10-7 ng/μL. Mouse models (Meriones unguiculatus) of Tyzzer disease were established and a LAMP-lateral flow dipstick (LAMP-LFD) was developed for detecting C. piliforme. The detection rate of C. piliforme was 5.08% in clean-grade animals and 9.96% in specific-pathogen-free-grade animals from Jiangsu, Zhejiang and Shanghai. In addition, the detection rates of C. piliforme were 10.1%, 8.6% and 20%, in animals from Hangzhou, Wenzhou and Shaoxing, respectively. The detection rate of C. piliforme was higher in experimental animals used in schools than in those used in companies and research institutes.ConclusionsThe LAMP-LFD method established in this study can be used to detect C. piliforme in animals handled in laboratory facilities of universities, pharmaceutical enterprises and research and development institutions.
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Project description:The spread of African swine fever virus (ASFV) caused huge economic costs, so early detection is particularly important. Here, we established a fluorescence biosensor based on carbon nanodots (CNDs) and loop-mediated isothermal amplification (LAMP) to ultra-sensitively detect ASFV. LAMP with high efficiency produced a large amount of pyro phosphoric acid and caused pH change in a short time. CNDs with strong light stability had a large fluorescence response at the emission wavelength of 585.5 nm to small pH change by the excitation wavelength of 550 nm. The biosensor realized "turn-off-on" mode for ASFV detection with the detection limit as low as 15.21 copies μL-1. In addition, the biosensor had high accuracy in the actual sample assay. Therefore, the biosensor achieved rapid, sensitive, low-cost, and simple detection for ASFV. Moreover, the biosensor broadened the detection pathway of LAMP as a tool with great development prospect.
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Project description:African Swine Fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and lethal viral disease of pigs. However, commercial vaccines are not yet available, and neither are drugs to prevent or control ASF. Therefore, rapid, accurate on-site diagnosis is urgently needed for detection during the early stages of ASFV infection. Herein, a cleaved probe-based loop-mediated isothermal amplification (CP-LAMP) detection method was established. Based on the original primer sets, we targeted the ASFV 9GL gene sequence to design a probe harboring a ribonucleotide insertion. Ribonuclease H2 (RNase H2) enzyme activity can only be activated when the probe is perfectly complementary, resulting in hydrolytic release of a quencher moiety, and consequent signal amplification. The method displayed robust sensitivity, with copy number detection as low as 13 copies/µL within 40 min at constant temperature (62°C). Visualization of the fluorescence product was employed using a self-designed 3D-printed visualization function cassette, and the CP-LAMP method achieved specific identification and visual detection of ASFV. Moreover, coupling the dual function cassette and smartphone quantitation makes the CP-LAMP assay first user-friendly, cost-effective, portable, rapid, and accurate point-of-care testing (POCT) platform for ASFV.
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Project description:African swine fever virus (ASFV), the etiological agent of African swine fever (ASF), a hemorrhagic fever of domestic pigs, has devastating consequences for the pig farming industry. More than 1,000,000 pigs have been slaughtered since 3 August 2018 in China. However, vaccines or drugs for ASF have yet to be developed. As such, a rapid test that can accurately detect ASFV on-site is important to the timely implementation of control measures. In this study, we developed a rapid test that combines recombinase polymerase amplification (RPA) of the ASFV p72 gene with lateral flow detection (LFD). Results showed that the sensitivity of recombinase polymerase amplification with lateral flow dipstick (RPA-LFD) for ASFV was 150 copies per reaction within 10 min at 38°C. The assay was highly specific to ASFV and had no cross-reactions with other porcine viruses, including classical swine fever virus (CSFV). A total of 145 field samples were examined using our method, and the agreement of the positive rate between RPA-LFD (10/145) and real-time PCR (10/145) was 100%. Overall, RPA-LFD provides a novel alternative for the simple, sensitive, and specific identification of ASFV and showed potential for on-site ASFV detection.
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Project description:Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP-ELISA) and with lateral flow dipstick (LAMP-LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP-ELISA and LAMP-LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. The optimum time and temperature for LAMP were 60 min and 65°C, respectively. The specific capture oligonucleotide probes, biotin-labeled CPV probe for LAMP-ELISA and fluorescein isothiocyanate-labeled CPV probe for LAMP-LFD were also designed for hybridization with LAMP amplicons on streptavidin-coated wells and LFD strips, respectively. For the comparison of detection sensitivity, conventional PCR and LAMP for CPV detection were also performed. The CPV detection limits by PCR, PCR-ELISA, LAMP, LAMP-ELISA and LAMP-LFD were 10(2), 10(2), 10(-1), 10(-1) and 10(-1) TCID50/ml, respectively. In tests using artificially contaminated dog fecal samples, the samples with CPV inoculation levels of ≥1 TCID50/ml gave positive results by both LAMP-ELISA and LAMP-LFD. Our data indicated that both LAMP-ELISA and LAMP-LFD are promising as rapid, sensitive and specific methods for an efficient diagnosis of CPV infection.
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