scholarly journals Lateral flow biosensor combined with loop-mediated isothermal amplification for detection of legionella pneumophila.

2021 ◽  
Author(s):  
Luxi Jiang ◽  
Xiaomeng Li ◽  
Rumeng Gu ◽  
Ziling Shi ◽  
Meijun Song ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Culture Method ◽  
Lavage Fluid ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Specific Gene ◽  
Minimum Concentration ◽  
Loop Mediated Isothermal Amplification ◽  
Whole Process

Abstract Legionella pneumophila ( L. pneumophila ) is the most pathogenic species of Legionella , which can cause Legionella disease. It can cause pneumonia, or Pontiac fever. In severe cases, it can lead to respiratory failure and kidney failure, with a high fatality rate. Here, a novel molecular diagnosis method, a loop-mediated isothermal amplification coupled with lateral flow biosensor (LFB) method (LAMP-LFB) was successfully established and evaluated for the identification of L. pneumophila . A set of 6 primers was designed specifically based on the L. pneumophila -specific gene mip. The optimized time and temperature conditions for the LAMP was 50 min and 64◦C respectively. The minimum concentration that can be detected by this method was 100fg. Using the protocol, we could observe the LAMP amplification within 2min by LFB. The whole process, including the preparation of DNA (20 min), LAMP reaction (50 min) and results reporting (2 min), could be finished within 75 min. Among 50 alveolar lavage fluid samples, 5(10%) were L. pneumophila -positive by the LAMP-LFB, and the diagnostic accuracy was 100% when compared to the culture method. While only 4 samples were positive using PCR method. In a word, the LAMP-LFB assay is a rapid, sensitive and specific detection method that can detect Legionella pneumophila , and it can be used as a new molecular method for the detection of target pathogens in water, environmental and clinical samples.

scholarly journals Rapid Detection of Aspergillus fumigatus Using Multiple Cross Displacement Amplification Combined With Nanoparticles-Based Lateral Flow

2021 ◽  
Vol 11 ◽  
Author(s):  
Luxi Jiang ◽  
Xiaomeng Li ◽  
Rumeng Gu ◽  
Deguang Mu
Keyword(s):  
Aspergillus Fumigatus ◽  
Detection Method ◽  
Culture Method ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Specific Detection ◽  
Minimum Concentration ◽  
Whole Process ◽  
Multiple Cross ◽  
Pcr Method

Aspergillus fumigatus is an opportunistic, ubiquitous, saprophytic mold which can cause infection in the lungs, nose, eyes, brain, and bones in humans, especially in immunocompromised patients. However, it is difficult to diagnose A. fumigatus infection quickly. Here, we introduce a new detection method, namely multiple cross displacement amplification (MCDA) combined with nanoparticle-based lateral flow biosensor (LFB) (MCDA-LFB), which was proved to be fast, reliable, and simple for detecting A. fumigatus. We designed a set of 10 primers targeting the gene annexin ANXC4 of A. fumigatus. The best MCDA condition is 66 °C for 35 min. The minimum concentration that can be detected by this method was 10 fg. In the case of 100 sputum samples, 20 (20%) and 15 (15%) samples were positive by MCDA-LFB and PCR method, respectively. MCDA-LFB and traditional culture method showed the same results. Compared with the culture method, the diagnostic accuracy of MCDA-LFB can reach 100%. It showed that the MCDA-LFB method has better detection ability than the PCR method. We found that the whole process could be controlled within 60 min including the preparation of DNA (20 min), MCDA reaction (35 min) and results reporting (2 min). These results show that this assay is suitable for the rapid, sensitive and specific detection of A. fumigatus in clinical samples.

scholarly journals Rapid and Visual Differentiation of Mycobacterium tuberculosis From the Mycobacterium tuberculosis Complex Using Multiplex Loop-Mediated Isothermal Amplification Coupled With a Nanoparticle-Based Lateral Flow Biosensor

2021 ◽  
Vol 12 ◽  
Author(s):  
Xinggui Yang ◽  
Junfei Huang ◽  
Xu Chen ◽  
Ziyu Xiao ◽  
Xiaojuan Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Limit Of Detection ◽  
Clinical Specimen ◽  
Lamp Assay ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Whole Process ◽  
Specimen Processing ◽  
Mycobacterium Africanum

Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis (MTB), but other members of the Mycobacterium tuberculosis complex (MTBC), especially Mycobacterium bovis (pyrazinamide-resistant organisms), may also be involved. Thus, the ability to rapidly detect and identify MTB from other MTBC members (e.g., M. bovis, Mycobacterium microti, Mycobacterium africanum) is essential for the prevention and treatment of TB. A novel diagnostic method for the rapid detection and differentiation of MTB, which employs multiplex loop-mediated isothermal amplification (mLAMP) combined with a nanoparticle-based lateral flow biosensor (LFB), was established (mLAMP-LFB). Two sets of specific primers that target the IS6110 and mtp40 genes were designed according to the principle of LAMP. Various pathogens were used to optimize and evaluate the mLAMP-LFB assay. The optimal conditions for mLAMP-LFB were determined to be 66°C and 40 min, and the amplicons were directly verified by observing the test lines on the biosensor. The LAMP assay limit of detection (LoD) was 125 fg per vessel for the pure genomic DNA of MTB and 4.8 × 103 CFU/ml for the sputum samples, and the analytical specificity was 100%. In addition, the whole process, including the clinical specimen processing (35 min), isothermal amplification (40 min), and result confirmation (1–2 min), could be completed in approximately 80 min. Thus, mLAMP-LFB is a rapid, reliable, and sensitive method that is able to detect representative members of MTBC and simultaneously differentiate MTB from other MTBC members, and it can be used as a potential screening tool for TB in clinical, field, and basic laboratory settings.

scholarly journals Rapid detection of Clostridium perfringens in food by loop-mediated isothermal amplification combined with a lateral flow biosensor

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245144
Author(s):  
Thanawat Sridapan ◽  
Wanida Tangkawsakul ◽  
Tavan Janvilisri ◽  
Wansika Kiatpathomchai ◽  
Sirintip Dangtip ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Culture Method ◽  
Food Samples ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Bacterial Strains ◽  
Commercial Test ◽  
Loop Mediated Isothermal Amplification ◽  
Test Kit

Clostridium perfringens is a key anaerobic pathogen causing food poisoning. Definitive detection by standard culture method is time-consuming and labor intensive. Current rapid commercial test kits are prohibitively expensive. It is thus necessary to develop rapid and cost-effective detection tool. Here, loop-mediated isothermal amplification (LAMP) in combination with a lateral-flow biosensor (LFB) was developed for visual inspection of C. perfringens-specific cpa gene. The specificity of the developed test was evaluated against 40 C. perfringens and 35 other bacterial strains, which showed no cross-reactivity, indicating 100% inclusivity and exclusivity. LAMP-LFB detection limit for artificially contaminated samples after enrichment for 16 h was 1–10 CFU/g sample, which was comparable to the commercial real-time PCR kit. The detection performance of LAMP-LFB was also compared to culture-based method using 95 food samples, which revealed the sensitivity (SE), specificity (SP) and Cohen's kappa coefficient (κ) of 88.0% (95% CI, 75.6%-95.4%), 95.5% (95% CI, 84.8%-99.4%) and 0.832 (95% CI, 0.721–0.943), respectively. Area under the receiver operating characteristic (ROC) curve was 0.918 (95% CI, 0.854–0.981), indicating LAMP-LFB as high relative accuracy test. In conclusion, LAMP-LFB assay is a low-cost qualitative method and easily available for routine detection of C. perfringens in food samples, which could serve as an alternative to commercial test kit.

Development and application of loop-mediated isothermal amplification label-based nanoparticles lateral flow biosensor for detection of Mycobacterium tuberculosis

2019 ◽  
Author(s):  
Xingyun Wang ◽  
Yi Wang ◽  
Weiwei Jiao ◽  
Guirong Wang ◽  
Yacui Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Diagnostic Technique ◽  
Cost Effective ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
High Morbidity ◽  
Analytical Sensitivity ◽  
Loop Mediated Isothermal Amplification

Abstract Tuberculosis is a serious disease with high morbidity and mortality, thus rapid and cost-effective diagnostic test for Mycobacterium tuberculosis (MTB) is urgently needed. Here, a novel detection diagnostic technique, termed as loop-mediated isothermal amplification label-based nanoparticles with lateral flow biosensor (LAMP-LFB), was developed and evaluated for rapid, reliable and objective detection of MTB. Two sets of primers, which targeted IS 6110 and IS 1081 sequences of MTB, were simultaneously designed for establishment of LAMP-LFB assay. The optimal reaction conditions of MTB-LAMP-LFB assay confirmed were 66ºC for only 50min. The analytical sensitivity of MTB-LAMP-LFB is 10fg of genomic templates in pure culture, and the detection results obtained from LFB was in conformity with agarose gel electrophoresis. No cross-reactivity with other common bacteria and non-tuberculous mycobacteria strains (NTM) was obtained. A total of 158 clinical samples were collected from presumptive 158 TB patients, were used for evaluating the feasibility of MTB-LAMP-LFB assay. Among 98 TB patients diagnosed with composite reference standard, the positive rate for MTB detection using liquid culture, Xpert MTB/RIF and LAMP-LFB were 40.0% (39/98), 50.0% (48/98), and 86.7% (85/98), respectively. Among 39 culture confirmed samples, 84.6% (33/39) cases were Xpert MTB/RIF-positive and 92.3% (36/39) were LAMP-LFB-positive. For the 59 clinically diagnosed TB cases 25.4% (15/59) and 83.0% (49/59) were Xpert MTB/RIF-positive and LAMP-LFB positive, respectively. Therefore, MTB-LAMP-LFB assay is a simple, reliable, and sensitive method for MTB detection and maybe prospective in early diagnosis of MTB.

scholarly journals Rapid and Visual Detection of SARS-CoV-2 Using Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Linked With Gold Nanoparticle-Based Lateral Flow Biosensor

2021 ◽  
Vol 11 ◽  
Author(s):  
Xu Chen ◽  
Qingxue Zhou ◽  
Shijun Li ◽  
Hao Yan ◽  
Bingcheng Chang ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Prevention And Control ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification ◽  
The World ◽  
And Control

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has caused the outbreak of coronavirus disease 2019 (COVID-19) all over the world. In the absence of appropriate antiviral drugs or vaccines, developing a simple, rapid, and reliable assay for SARS-CoV-2 is necessary for the prevention and control of the COVID-19 transmission.MethodsA novel molecular diagnosis technique, named multiplex reverse transcription loop-mediated isothermal amplification, that has been linked to a nanoparticle-based lateral flow biosensor (mRT-LAMP-LFB) was applied to detect SARS-CoV-2 based on the SARS-CoV-2 RdRp and N genes, and the mRT-LAMP products were analyzed using nanoparticle-based lateral flow biosensor. The mRT-LAMP-LFB amplification conditions, including the target RNA concentration, amplification temperature, and time were optimized. The sensitivity and specificity of the mRT-LAMP-LFB method were tested in the current study, and the mRT-LAMP-LFB assay was applied to detect the SARS-CoV-2 virus from clinical samples and artificial sputum samples.ResultsThe SARS-CoV-2 specific primers based on the RdRp and N genes were valid for the establishment of mRT-LAMP-LFB assay to detect the SARS-CoV-2 virus. The multiple-RT-LAMP amplification condition was optimized at 63°C for 30 min. The full process, including reaction preparation, viral RNA extraction, RT-LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the mRT-LAMP-LFB technology was 20 copies per reaction. The specificity of mRT-LAMP-LFB detection was 100%, and no cross-reactions to other respiratory pathogens were observed.ConclusionThe mRT-LAMP-LFB technique developed in the current study is a simple, rapid, and reliable method with great specificity and sensitivity when it comes to identifying SARS-CoV-2 virus for prevention and control of the COVID-19 disease, especially in resource-constrained regions of the world.

scholarly journals Detection ofMycobacterium tuberculosisby Using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick in Clinical Samples

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Thongchai Kaewphinit ◽  
Narong Arunrut ◽  
Wansika Kiatpathomchai ◽  
Somchai Santiwatanakul ◽  
Pornpun Jaratsing ◽  
...  
Keyword(s):  
Communicable Disease ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Lamp Method ◽  
Cross Hybridization ◽  
Persistent Problem ◽  
Loop Mediated Isothermal Amplification ◽  
Lateral Flow Dipstick ◽  
Epidemiological Surveys

Tuberculosis (TB) is a communicable disease caused by the bacteriumMycobacterium 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 IS6110gene ofM. tuberculosisspecifically 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 detectM. tuberculosisgenomic DNA as little as 5 pg. The technique showed a significant specificity since no cross-hybridization toM. intracellulare(MIC),M. fortuitum(MFT),M. avium(MAV),M. kansasii(MKS), andM. 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.

scholarly journals Lateral Flow Loop-Mediated Isothermal Amplification Test with Stem Primers: Detection ofCryptosporidiumSpecies in Kenyan Children Presenting with Diarrhea

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Timothy S. Mamba ◽  
Cecilia K. Mbae ◽  
Johnson Kinyua ◽  
Erastus Mulinge ◽  
Gitonga Nkanata Mburugu ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Flow Loop ◽  
Loop Mediated Isothermal Amplification ◽  
Higher Sensitivity

Background. Cryptosporidiumis a protozoan parasite and a major cause of diarrhea in children and immunocompromised patients. Current diagnostic methods for cryptosporidiosis such as microscopy have low sensitivity while techniques such as PCR indicate higher sensitivity levels but are seldom used in developing countries due to their associated cost. A loop-mediated isothermal amplification (LAMP) technique, a method with shorter time to result and with equal or higher sensitivity compared to PCR, has been developed and applied in the detection ofCryptosporidiumspecies. The test has a detection limit of 10 pg/µl (~100 oocysts/ml) indicating a need for more sensitive diagnostic tools. This study developed a more sensitive lateral flow dipstick (LFD) LAMP test based on SAM-1 gene and with the addition of a second set of reaction accelerating primers (stem primers).Results. The stem LFD LAMP test showed analytical sensitivity of 10 oocysts/ml compared to 100 oocysts/ml (10 pg/ul) for each of the SAM-1 LAMP test and nested PCR. The stem LFD LAMP and nested PCR detected 29/39 and 25/39 positive samples of previously identifiedC. parvumandC. hominisDNA, respectively. The SAM-1 LAMP detected 27/39. On detection ofCryptosporidiumDNA in 67 clinical samples, the stem LFD LAMP detected 16 samples and SAM-2 LAMP 14 and nested PCR identified 11. Preheating the templates increased detection by stem LFD LAMP to 19 samples. Time to results from master mix preparation step took ~80 minutes. The test was specific, and no cross-amplification was recorded with nontarget DNA.Conclusion.The developed stem LFD LAMP test is an appropriate method for the detection ofC. hominis, C. parvum,andC. meleagridisDNA in human stool samples. It can be used in algorithm with other diagnostic tests and may offer promise as an effective diagnostic tool in the control of cryptosporidiosis.

scholarly journals Development and Preliminary Application of Multiplex Loop-Mediated Isothermal Amplification Coupled With Lateral Flow Biosensor for Detection of Mycobacterium tuberculosis Complex

2021 ◽  
Vol 11 ◽  
Author(s):  
Xingyun Wang ◽  
Guirong Wang ◽  
Yacui Wang ◽  
Shuting Quan ◽  
Hui Qi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Tuberculosis Complex ◽  
Fluorescein Isothiocyanate ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Tuberculosis Complex ◽  
Loop Mediated Isothermal Amplification

The aim of this study was to develop a simple and reliable method to detect Mycobacterium tuberculosis complex (MTBC) and verify its clinical application preliminarily. A loop-mediated isothermal amplification method coupled with lateral flow biosensor (LAMP-LFB) assay, was developed and evaluated for detection of MTBC. Two sets of primers, which targeted IS6110 and IS1081 sequences of MTBC, were designed for establishment of multiplex LAMP-LFB assay. The amplicons were labelled with biotin and fluorescein isothiocyanate (FITC) by adding FITC labelled primer and biotin-14-dATP and biotin-14-dCTP and could be visualized using LFB. The optimal reaction conditions of multiplex LAMP-LFB assay confirmed were 66°C for 50 min. The analytical sensitivity of multiplex LAMP-LFB is 10 fg of genomic templates using pure culture, and no cross-reactivity with other common bacteria and non-tuberculous mycobacteria strains was obtained. A total of 143 clinical samples collected from 100 TB patients (62 definite TB cases and 38 probable TB cases) and 43 non-TB patients were used for evaluating the feasibility of multiplex LAMP-LFB assay. The multiplex LAMP-LFB (82.0%, 82/100) showed higher sensitivity than culture (47.0%, 47/100, P < 0.001) and Xpert MTB/RIF (54.0%, 54/100, P < 0.001). Importantly, the multiplex LAMP-LFB assay detected additional 28 probable TB cases, which increased the percentage of definite TB cases from 62.0% (62/100) to 90.0% (90/100). The specificity of multiplex LAMP-LFB assay in patients without TB was 97.7% (42/43). Therefore, multiplex LAMP-LFB assay is a simple, reliable, and sensitive method for MTBC detection, especially in probable TB cases and resource limited settings.

scholarly journals Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis

Diagnostics ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 32
Author(s):  
Supriya Sharma ◽  
Sandeep Kumar ◽  
Md.Zohaib Ahmed ◽  
Nitin Bhardwaj ◽  
Jaskirat Singh ◽  
...  
Keyword(s):  
Point Of Care ◽  
Melting Curve ◽  
Lateral Flow ◽  
Curve Analysis ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Melting Curve Analysis ◽  
Loop Mediated Isothermal Amplification ◽  
Malaria Species ◽  
Flow Detection

Isothermal techniques with lateral flow detection have emerged as a point of care (POC) technique for malaria, a major parasitic disease in tropical countries such as India. Plasmodium falciparum and Plasmodium vivax are the two most prevalent malaria species found in the country. An advanced multiplex loop-mediated isothermal amplification (mLAMP) combined with a lateral flow dipstick (LFD) technique was developed for the swift and accurate detection of P. falciparum and P. vivax, overcoming the challenges of the existing RDTs (rapid diagnostic tests). A single set of LAMP primers with a biotinylated backward inner primer (BIP primer) was used for DNA amplification of both malaria species in a single tube. The amplified DNA was hybridized with fluorescein isothiocyanate (FITC) and digoxigenin-labelled DNA probes, having a complemented sequence for the P. falciparum and P. vivax genomes, respectively. A colour band appeared on two separate LFDs for P. falciparum and P. vivax upon running the hybridized solution over them. In total, 39 clinical samples were collected from ICMR-NIMR, New Delhi. Melting curve analysis, with cross primers for both species, was used to ascertain specificity, and the sensitivity was equated with a polymerase chain reaction (PCR). The results were visualized on the LFD for both species within 60 min. We found 100% sensitivity and specificity, when compared with a traditional PCR. Melting curve analysis of mLAMP revealed the lowest detection limit of 0.15 pg/μL from sample genomic DNA. The mLAMP-LFD assays could be a potential point of care (POC) tool for early diagnosis in non-laboratory conditions, with the convenience of a reduced assay time and the simple interpretation of results.

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