Visual Loop-mediated Isothermal Amplification Research Articles

Visual LAMP Method for Detection of White Spot Syndrome Virus in Shrimp and Crayfish

White spot syndrome virus (WSSV) has caused large economic losses to the aquaculture industry, and thus far there are no efficient therapeutic treatments available against this lethal virus. Therefore, rapid detection of white spot syndrome virus (WSSV) in shrimp is crucial. This study was conducted to establish a rapid visual detection method for WSSV in shrimp and on the basis of loop-mediated isothermal amplification (LAMP). The color change of the amplification products was observed using a fluorescence amplification curve (with the SYTO-9 fluorescent dye) and LAMP visualization (with calcein), and the method`s specificity, sensitivity, and reproducibility were analyzed. It showed that this method can specifically detect WSSV, with a detection sensitivity of 1 fg/μL and has good reproducibility and reliability. This method was used to analyze cultured shrimp and crayfish samples; among 329 suspected clinical samples, the positive diagnostic rate of WSSV was 27.96%. The infection activity of WSSV was the highest at temperatures of 20–26°C. The LAMP-based method for rapid visual detection of WSSV does not need complex experimental instruments, and an equipment with a stable heat source is sufficient for the reaction. It is easy to use and can be used at shrimp breeding sites.

Rapid on-site identification of Lepiota brunneoincarnata-induced mushroom poisoning by simple DNA extraction and loop-mediated isothermal amplification strategy

Lepiota brunneoincarnata-induced mushroom poisoning has occurred widely in some areas of the world in recent years, resulting in high mortality and posing potential health risks to humans and pets. However, a rapid and sensitive method for the identification of L. brunneoincarnata is scarce. Loop-mediated isothermal amplification (LAMP) combined with visual dye reaction or lateral flow dipstick (LFD) was established for rapid on-site detection of L. brunneoincarnata. Simple DNA extraction was completed by the heating lysis method within 15 min. In addition, a specific LAMP primer set was designed targeting the internal transcribed spacer (ITS) sequence of L. brunneoincarnata, with no cross-reactivity against 22 other mushroom species. Sensitivity evaluation showed that the detection limits of visual LAMP and LAMP-LFD assays were 10 and 0.1 pg/μL genomic DNA of L. brunneoincarnata, respectively. Moreover, the developed method was successfully applied to cooked mushroom and simulated vomitus, with detection limits of 1% admixture for the visual LAMP assay and 0.1% admixture for the LAMP-LFD assay. The whole detection protocol can be completed in 60 min without expensive instruments, which is suitable for the rapid identification of Lepiota brunneoincarnata-induced mushroom poisoning in the field.

Rapid Russula senecis identification assays using loop-mediated isothermal amplification based on real-time fluorescence and visualization.

Russula senecis, a common poisonous mushroom, is widely distributed in China. Mushroom poisoning is becoming a major threat to human health and its rate is increasing worldwide. For the first time, we developed a set of loop-mediated isothermal amplification (LAMP) assays based on a real-time fluorescence and a visualization method to detect R. senecis, and the visual LAMP reaction system was optimized to further shorten the reaction time. Both real-time LAMP and visual LAMP could detect as low as 3.2pg of genomic DNA. In addition, fried and digested mushrooms were used to validate the proposed LAMP method, and mushroom mixtures with as low as 1% of the target species could be successfully detected, indicating that the LAMP assays established in this study had good applicability and could be used for clinical sample detection and forensic identification. Furthermore, the LAMP assays were proven to be comparable to the real-time PCR method. KEY POINTS: • A set of loop-mediated isothermal amplification (LAMP) assays based on real-time fluorescence and visualization to detect Russula senecis was developed. • Both real-time LAMP and visual LAMP can be used to detect genomic DNA at concentrations as low as 3.2pg. • By simulating mushroom processing and digestion in gastric juice, LAMP assays were proved to have good applicability and could be used for clinical diagnosis and forensic analysis.

Rapid identification of Takifugu genus using visual loop-mediated isothermal amplification.

Some Takifugu species are commonly found in the coastal areas of China, Japan, Thailand, and Korea and cause pufferfish poisoning, which is toxic and even lethal to humans. From 2010 to 2015, there were 430 cases of pufferfish poisoning worldwide, resulting in 52 deaths. Identification of Takifugu species is imperative to reduce financial losses and ensure food safety. Here, visual loop-mediated isothermal amplification (LAMP) was applied to identify Takifugu species. Conserved regions within the mitochondrial DNA among different Takifugu species were selected to design LAMP primers. In 55min of amplification, sufficient DNA was obtained to observe the results with the naked eye, without the need for complicated instruments. The method was highly specific, with no cross-detection of 17 other fish species, namely, 7 Tetraodontiformes species and 10 commercially important fish. The method showed a detection limit of 0.1ng Takifugu DNA and was successfully validated to detect Takifugu in cooked fish and the vomitus of poisoned patients. This rapid and visual LAMP method is a useful tool to prevent false labeling, protect consumer rights, and reduce the risk of pufferfish poisoning. PRACTICAL APPLICATION: The loop-mediated isothermal amplification method established in this study can identify cooked or digested fish products containing 1% or more of Takifugu. Therefore, it can be used for the visual detection of Takifugu products and the medical diagnosis of Takifugu poisoning.

Development and Clinical Validation of a Potential Penside Colorimetric Loop-Mediated Isothermal Amplification Assay of Porcine Circovirus Type 3.

Porcine circovirus type 3 (PCV3), a novel circovirus, imposes great burdens on the global pig industry. The penside tests for detecting PCV3 are critical for assessing the epidemiological status and working out disease prevention and control programs due to the unavailability of a commercial vaccine. A one-step molecular assay based on visual loop-mediated isothermal amplification (vLAMP) was developed for simple and rapid detection of PCV3. We compared its sensitivity and specificity with TaqMan quantitative real-time polymerase chain reaction (qPCR) and applied the developed assay in the epidemiological study of (n = 407) pooled swine sera collected from almost the entire mainland China during the years 2017–2018. We also explored the feasibility of the vLAMP assay for detecting raw samples without a prior DNA isolation step to expand its application capability. Results showed that the vLAMP assay could reliably detect the PCV3 cap gene with a detection limit of 10 DNA copies equal to that of the Taqman qPCR assay. In the epidemiological study, the PCV3 positive detection rate for 407 swine pooled sera detected by the vLAMP assay was 37.35% (152/407), whereas it was 39.01% (159/407) for Taqman qPCR. For the detection method without genome extraction, the results kept satisfactory specificity (100%) but displayed lower sensitivity (100% for CT < 32), indicating the direct detection is not sensitive enough to discriminate the samples with low viral loads. The one-step vLAMP is a convenient, rapid, and cost-effective diagnostic for penside detection and will enable the epidemiological surveillance of PCV3, which has widely spread in mainland China.

Rapid identification of Hebeloma crustuliniforme species using real-time fluorescence and visual loop-mediated isothermal amplification based on the internal transcribed spacer sequence

Mushroom poisoning is responsible for nearly 50% of all oral poisoning-related deaths in China. Most mushroom poisoning incidences are caused by the difficulty in distinguishing whether wild mushrooms are poisonous or not. In this study, we aimed to develop two rapid and sensitive methods for Hebeloma crustuliniforme identification using visual and real-time loop-mediated isothermal amplification (LAMP) technology. LAMP primers were designed targeting the Hebeloma crustuliniforme internal transcribed spacer (ITS) sequence and their specificity was tested against 41 other mushroom species. We found that the LAMP primers could specifically recognize the Hebeloma crustuliniforme ITS sequence without cross reacting with that of other mushrooms. Both LAMP methods could detect as low as 1 pg/μL genomic DNA and 0.01% Hebeloma crustuliniforme in different mushroom mixtures. The whole detection process was completed within 35 min, with no requirement of complicated instruments for performing visual LAMP. The two methods established in this study can not only be used for the identification of fresh mushrooms but also for cooked mushrooms and mushrooms digested by gastric juice. To the best of our knowledge, this is the first study to establish a detection method for Hebeloma crustuliniforme identification. This rapid, sensitive, and visual method could be used on-site for poisonous mushroom detection and prevention of possible food poisoning incidents.

Rapid and Sensitive Detection of the Colistin Resistance Gene mcr-3 by Loop-Mediated Isothermal Amplification and Visual Inspection

Objective: The distribution of colistin resistance in mcr-carrying bacteria poses a threat to global public health. In particular, the newly identified mcr-3 allele has spread globally, especially in China, second only to mcr-1. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid, sensitive, and visual detection of the presence of the mcr-3 gene. Materials and Methods: A total of 13 clinical bacterial strains and 11 negative strains were used in this study. We designed LAMP Primers, optimized reaction conditions, used three different methods to detect LAMP amplification products: (1) agarose gel electrophoresis, (2) LAMP-hydroxy naphthol blue (HNB) detection, (3) LAMP-SYBR Green I (LAMP-SGI) visual inspection, and evaluated its specificity and sensitivity. Results: The amplification reaction was completed in 1 hr at 62°C under isothermal conditions. The final optimized mixtures contained 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100, 1.2 μL HNB, and 0.5 μL SYBR Green I as additives to the initial reaction mixture. LAMP detection, including two visual methods, LAMP-HNB and LAMP-SGI, of mcr-3 possessed the same specificity and a 10-fold higher sensitivity compared with a conventional polymerase chain reaction assay using the same samples. Conclusion: We successfully established an mcr-3 LAMP detection with portability and rapidity of the reaction by the easily distinguishable color changes in the reaction tubes. This visual LAMP assay for mcr-3 detection was simple, time saving, and economical, especially suited to field laboratories.

Closed-tube field-deployable loop-mediated isothermal amplification (LAMP) assay based on spore wall protein (SWP) for the visual detection of Enterocytozoon hepatopenaei (EHP)

Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment.

Design of Rapid Detection System for Five Major Carbapenemase Families (bla KPC, bla NDM, bla VIM, bla IMP and bla OXA-48-Like) by Colorimetric Loop-Mediated Isothermal Amplification.

PurposeCarbapenemase-producing Enterobacteriaceae (CPE) infection constitutes a public health threat. Timely and efficient diagnosis is of paramount importance for prompt and effective therapy. In order to quickly and comprehensively detect the five major families of carbapenemases (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48-like), colorimetric loop-mediated isothermal amplification (LAMP) was employed.Materials and MethodsFive sets of LAMP primers were designed, each of which can, respectively, amplify all the carbapenemase subtypes described in this work. Twenty whole genome sequencing-verified-“standard strains”, including 1 blaNDM-1, 1 blaNDM-5, 1 blaNDM-6, 1 blaNDM-7, 2 blaIMP-4, 1 blaIMP-8, 2 blaKPC-2, 1 blaKPC-3, 1 blaKPC-4, 1 blaKPC-5, 1 blaKPC-6, 1 blaKPC-7, 1 blaOXA-48 and 1 blaOXA-181 carrier, and 1 blaVIM and blaOXA-244, 1 blaKPC-2 and blaIMP-4, 1 blaKPC-2 and blaVIM-1 and 1 blaKPC-2 and blaNDM-1-co-carriers, were used to establish a 25-microliter visual LAMP reaction system (kept at 65°C for 30 minutes in water bath). Color change from bright pink to yellow indicated positive amplification. In addition, 126 pre-verified clinical carbapenem-resistant Enterobacteriaceae (CRE) isolates, including 65 CPE (23 blaNDM, 2 blaOXA-48-like, 1 blaKPC and blaVIM, 2 blaIMP, and 37 blaKPC carriers) and 61 non-CPE, were also detected.ResultsWith the lowest detection limit of 10 colony forming units (CFU) per reaction for LAMP and 103 CFU per reaction for PCR, the LAMP system demonstrated dramatically higher sensitivity while retaining the same specificity. Furthermore, we demonstrated concordant results between the two methods for the 126 clinical isolates.ConclusionTherefore, LAMP could be used for rapid identification of the five major carbapenemase gene families in routine clinical laboratories.

Development of a Potential Penside Colorimetric LAMP Assay Using Neutral Red for Detection of African Swine Fever Virus.

African swine fever (ASF) has caused huge economic losses to the swine industry worldwide. Since there is no commercial ASF vaccine available, an early diagnosis is extremely important to prevent and control the disease. In this study, ASF virus (ASFV) capsid protein-encoding gene (p72) was selected and used to design primers for establishing a one-step visual loop-mediated isothermal amplification (LAMP) assay with neutral red, a pH-sensitive dye, as the color shift indicator. Neutral red exhibited a sharp contrast of color change from faint orange (negative) to pink (positive) during LAMP for detection of ASFV. The designed primer set targeting highly conserved region of the p72 gene was highly specific to ASFV and showed no cross-reactivity with other swine viruses. The detection limit for the one-step visual LAMP developed was 10 copies/reaction based on the recombinant plasmid containing the p72 gene of ASFV. More importantly, the developed one-step visual LAMP showed high consistency with the results of the real-time polymerase chain reaction (qPCR) method recommended by World Organization for Animal Health (OIE). Furthermore, the results demonstrate that the colorimetric detection with this LAMP assay could be directly applied for the whole blood and serum samples without requiring genome extraction. Based on our results, the developed one-step visual LAMP assay is a promising penside diagnostic tool for development of early and cost-effective ASF monitoring program that would greatly contribute to the prevention and control of ASF.

Real-Time Fluorimetry Loop-Mediated Isothermal Amplification for Diagnosis of Leishmaniasis and as a Tool for Assessment of Cure for Post-Kala-Azar Dermal Leishmaniasis.

Despite the dwindling number of visceral leishmaniasis (VL) cases in India, there is an urgent need for early and unequivocal diagnostics for controlling and preventing the reemergence of VL. Post-kala-azar dermal leishmaniasis (PKDL), a dermal sequela of VL, serves as a reservoir of the parasite. Diagnosis of PKDL, especially the macular variant, is challenging and poses impediment toward attainment of VL elimination. In this study, a real-time fluorimetry loop-mediated isothermal amplification (RealAmp) assay has been established for the detection of different clinical manifestations of leishmaniasis. The study included 150 leishmaniasis patients (25 VL, 25 cutaneous leishmaniasis [CL], and 100-PKDL) along with 120 controls. The assay demonstrated sensitivity of 100% (95% CI: 86.68-100) for diagnosis of VL and PKDL (95% CI: 79.61-100) and 96% (95% CI: 86.68-100) for CL with 100% specificity. Moreover, considering the cardinal role of PKDL, diagnosis using minimally invasive slit aspirate was explored, which demonstrated remarkable sensitivity of 96% (95% CI: 87.64-98.47). As a test of cure for PKDL, RealAmp successfully detected parasite in two of posttreatment cases who later reported relapse on follow-up. Also, direct sample lysis using slit aspirate was attempted in a small group that yielded sensitivity of 89% (95% CI: 67.20-96.90). RealAmp depicted excellent diagnostic accuracy in the diagnosis of leishmaniasis in concordance with the established SYBR Green I-based (Molecular Probes, Eugene, OR) visual loop-mediated isothermal amplification (LAMP) and the reference comparator real-time PCR. The study endorsed the employment of LAMP either as visual-LAMP or RealAmp for an accurate and expeditious diagnosis of PKDL and as a tool for assessment of cure.

Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass

The red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

Real-time and visual detection of Cercospora canescens based on the cytb gene using loop-mediated isothermal amplification (LAMP)

Cercospora canescens Ellis & Martin is an important fungal pathogen that causes leaf spot and defoliation in mungbean (Vigna radiata (Linn.) Wilczek.). Molecular methods for detection of this pathogen have not yet been developed. In this study, we developed a real-time and visual loop-mediated isothermal amplification (LAMP) assay based on the cytochrome b (cytb) gene to detect C. canescens in mungbean. No cross-reactions were observed in specificity tests conducted with 10 other plant pathogenic fungal species. The minimum detectable concentration was 100 pg/μL C. canescens genomic DNA, and the amplified product could be visualized with the addition of SYBR Green I fluorescent dye, which could make the assay more convenient for deployment in the field. The LAMP method presented provides a specific, rapid and visual diagnostic tool for the identification of C. canescens, and has the potential to be standardized as a detection method for this pathogen in the field.

Molecular Identification of Anoplophora glabripennis (Coleoptera: Cerambycidae) From Frass by Loop-Mediated Isothermal Amplification

Anoplophora glabripennis (Motschulsky, 1853), native to eastern Asia, is a destructive woodborer of many ornamental species, leading to the decline and the death of the attacked trees. In outbreak areas as Europe or North America, this pest is usually identified using morphological or molecular analyses of adult or larval specimens. However, the procedures for collecting A. glabripennis specimens from infested plants are too expensive and time consuming for routine screening. A noninvasive diagnostic tool based on frass discrimination is therefore crucial for the rapid identification of A. glabripennis at different development stages in the host. This article describes a rapid diagnostic protocol based on loop-mediated isothermal amplification (LAMP). DNA extracted from A. glabripennis frass was amplified with both visual and real-time LAMP and compared with those of nontarget species. The results show that the method is reliable and accurate and therefore could be a promising diagnostic tool in phytosanitary surveys.

Visual closed-tube loop-mediated isothermal amplification (LAMP) assay targeting aerolysin gene: a practical screening method for virulent Aeromonas species affecting cultured eels in China

Under intensive and high-density aquaculture conditions, the cultured eels are easily infected by a variety of virulent Aeromonas species, which leads to high mortality rates and economic losses. In this study, a visual closed-tube loop-mediated isothermal amplification (LAMP) assay was developed for targeting aerolysin gene of Aeromonas species affecting cultured eels. Firstly, two pairs of outer and inner primers were designed against a conserved region of the aer gene from different Aeromonas species. Secondly, the weakly buffered aer-LAMP assay was optimized under various Mg2+ concentrations, different amplification times and amplification temperatures followed by the visual evaluation of phenol red dye and SYTO-9. Thirdly, the detection limit of the aer-LAMP assay showed 16.8 copy/μl which was simultaneously evaluated by the color changes from red to yellow and the real-time fluorescence amplification curves. The specificity of the aer-LAMP assay showed no cross-reaction with other related species. In addition, 30 Aeromonas strains and 40 clinical tissues from eels were used to further validate the effectiveness of the aer-LAMP assay. All the results showed that the aer-LAMP assay provided a rapid, simple, and reliable method for detecting Aeromonas harboring the aerolysin gene, which would be helpful for the prevention and control of Aeromonas infection in cultured eels.

Visual detection of high-risk HPV16 and HPV18 based on loop-mediated isothermal amplification

Increased adoption of HPV testing is expected in light of a growing global awareness of women's health and the recent launch of cervical cancer vaccines. Testing approaches must be both easy to implement, and offer intelligible representation of amplification results. Here, we introduce a simple, rapid, and visual HPV detection method based on a loop-mediated isothermal amplification (LAMP) assay. The visual LAMP assay can be successfully applied to amplify and differentiate the high-risk samples of HPV16 and HPV18, and results can be discriminated via the naked eye, without costly specialized apparatus. This HPV testing method has been evaluated with clinical samples and exhibited excellent reliability, as verified by polymerase chain reaction-microchip electrophoresis (PCR-MCE), and has a reduced false negative rate compared with cytological methods. The LAMP-based platforms with their facile operation and visual results possess great potential for on-site cervical cancer monitoring.

Construct-Specific Loop-Mediated Isothermal Amplification: Rapid Detection of Genetically Modified Crops with Insect Resistance or Herbicide Tolerance.

Insect resistant and herbicide tolerant genetically modified (GM) events have been approved in many countries. Screening methods could facilitate preliminary testing to check the GM status, which may target control elements, transgenes, and marker genes or construct regions. Among these, methods targeting the construct region, i.e., the junction between two genetic elements of a transgenic cassette are more specific. Loop-mediated isothermal amplification (LAMP) assays targeting three construct regions were developed; between Cauliflower Mosaic Virus 35S promoter and cry1Ac gene (p35S-cry1Ac), cry2Ab2 gene and nos terminator (cry2Ab2-tnos), and cp4-epsps gene and nos terminator (cp4epsps-tnos). LAMP assays were performed by incubation at constant temperatures for selected targets. Positive amplification was detected as a change in color from orange to green on addition of SYBR® Green dye in visual LAMP and as real-time amplification curves in real-time LAMP. These assays showed acceptable specificity and sensitivity. Visual LAMP was found to be sensitive enough to detect as low as 0.005%, equivalent to two target copies. Real-time LAMP assays were able to detect as low as four copies of the target within 40 min, making them suitable for rapid on-site testing for GM organisms (GMO). Practical utility was also verified using spiked test samples. These assays could be employed to address some of the biosafety or post-release monitoring issues, as well as to check for approved and unapproved GM events in a country. LAMP assays targeting three construct regions have been developed, enabling screening for approved or unapproved GMO.

Development and Standardization of Visual Loop Mediated Isothermal Amplification (LAMP) Essay for Specific Diagnosis of Johne’s Disease

Mycobacterium avium subspecies paratuberculosis (MAP), causative agent of Johne’s disease (JD) is chronic granulomatous enteritis affecting domestic and wild ruminants. Since, MAP is not killed by pasteurization, it has been isolated from commercially pasteurized milk and milk products resulting exposure of human population to this pathogen through milk. Control and eradication of JD is considered difficult because of its insidious nature and lack of early, rapid and accurate diagnostic tests. Therefore in present study, a visual loop-mediated isothermal amplification (LAMP) assay method has been developed using a total of six primers including 2 outer (F3 and B3), 2 inner (FIP and BIP) and 2 loop (LF and LB) primers specific for MAP for the first time on ‘S 5’ strain of Mycobacterium avium subsp. paratuberculosis ‘Indian Bison type’ biotype. After laboratory standardization, final optimized reaction performed at 65°C for 45 min was achieved after titration of incubation time, temperature conditions and the reporter dye calcein. Sensitivity and specificity of the LAMP assay was optimized and compared with traditional IS900 PCR. The sensitivity of LAMP assay was found to detect 10fg (100%) of DNA and 95.7% specificity was recorded with respect to traditional IS900 PCR. Comparison showed that LAMP had 98.6% and 96.1% sensitivity and specificity of 96.1% and 92.3%, with respect to microscopy and culture exhibiting ‘Almost perfect’ strength of agreement. The study concluded that LAMP assay was a reliable and sensitive diagnostic test to detect MAP infection in feces and can also be used for the ‘mass screening’ of the milk samples with the help of less expertise.

Visual detection of donkey-derived ingredients by loop-mediated isothermal amplification with 4-(2-pyridylazo)-resorcinol sodium salt

ABSTRACT The objective of this study was to develop a reliable visual method for rapid detection of donkey components to protect consumers from commercial adulteration. Six specific loop-mediated isothermal amplification (LAMP) primers targeted at donkey mitochondrial cytochrome b gene were designed, the mitochondrial DNA extraction simplified, the LAMP reaction system optimized, the specificity verified with mitochondrial DNA of horse, pork, cow, sheep, chicken, duck, and rabbit as negative controls, the detection limit determined with gradient dilution of adulterated meat with donkey meat, and the visual LAMP method for detection of donkey-derived ingredient in common meat products established. The results showed that the modified mitochondrial DNA extraction method was simple and repeatable, and the visual LAMP method with 4-(2-pyridylazo)-resorcinol sodium salt as indicator can accurately and specifically detect the donkey meat in common meat products, 1% detection limit. The study provided a promising solution for facilitating the surveillance of the commercial adulteration in processed meat.

Mini-Disk Capillary Array Coupling with LAMP for Visual Detection of Multiple Nucleic Acids using Genetically Modified Organism Analysis as an Example

Convenient, portable, and low-cost multiplex nucleic acid testing (NAT) systems are the trends in the fields of food safety, environmental microorganisms, molecular diagnosis, etc. In this study, we developed a novel system for visual monitoring of multiple nucleic acids combining a mini-disk capillary array (diameter = 17 mm, embedded with 6-10 capillaries), visual loop-mediated isothermal amplification (LAMP), and quick DNA extraction called mDC-LAMP. The performance and applicability of mDC-LAMP in testing multiple nucleic acids were evaluated and verified employing genetically modified contents analysis as an example. All of the results confirmed that mDC-LAMP has the advantages of high specificity without any cross contamination, high sensitivity with a limit of detection of 25 copies/reaction, high throughput with flexible channel sensors, easy fabrication, and low costs. We believe that mDC-LAMP is a competitive choice for on-spot monitoring of multiple nucleic acids in terms of the easy fabrication/operation, low costs, and suitable performance presented in the nucleic acids test.