A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to rapidly detect foot-and-mouth disease virus serotype C (FMDV C). By testing 10-fold serial dilutions of FMDV C samples, sensitivity of the FMDV C RT-LAMP was found to be 10 times higher than that of conventional reverse transcription-PCR (RT-PCR). No cross-reactivity with A, Asia 1, or O FMDV or swine vesicular disease virus (SVDV) indicated that FMDV C RT-LAMP may be an exciting novel method for detecting FMDV C.
Animals ; Foot-and-Mouth Disease/*diagnosis ; Foot-and-Mouth Disease Virus/genetics ; Nucleic Acid Amplification Techniques/*methods/veterinary ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Reverse Transcription/genetics ; Sensitivity and Specificity

Ascidian soft tunic syndrome (AsSTS) caused by Azumiobodo hoyamushi (A. hoyamushi) is a serious aquaculture problem that results in mass mortality of ascidians. Accordingly, the early and accurate detection of A. hoyamushi would contribute substantially to disease management and prevention of transmission. Recently, the loop-mediated isothermal amplification (LAMP) method was adopted for clinical diagnosis of a range of infectious diseases. Here, the authors describe a rapid and efficient LAMP-based method targeting the 18S rDNA gene for detection of A. hoyamushi using ascidian DNA for the diagnosis of AsSTS. A. hoyamushi LAMP assay amplified the DNA of 0.01 parasites per reaction and detected A. hoyamushi in 10 ng of ascidian DNA. To validate A. hoyamushi 18S rDNA LAMP assays, AsSTS-suspected and non-diseased ascidians were examined by microscopy, PCR, and by using the LAMP assay. When PCR was used as a gold standard, the LAMP assay showed good agreement in terms of sensitivity, positive predictive value (PPV), and negative predictive value (NPV). In the present study, a LAMP assay based on directly heat-treated samples was found to be as efficient as DNA extraction using a commercial kit for detecting A. hoyamushi. Taken together, this study shows the devised A. hoyamushi LAMP assay could be used to diagnose AsSTS in a straightforward, sensitive, and specific manner, that it could be used for forecasting, surveillance, and quarantine of AsSTS.
Animals ; Euglenozoa Infections/diagnosis/veterinary ; Kinetoplastida/*classification/genetics/*isolation & purification ; Nucleic Acid Amplification Techniques/*methods ; Predictive Value of Tests ; RNA, Ribosomal, 18S/*genetics ; Sensitivity and Specificity ; Urochordata

Animals ; Bacterial Vaccines ; Brucella suis ; genetics ; Brucellosis ; epidemiology ; microbiology ; veterinary ; China ; epidemiology ; DNA Fingerprinting ; DNA, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Nucleic Acid Amplification Techniques ; methods ; Time Factors

A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on T m values of 85.03 +/- 0.54degrees C for stx1 and 87.47 +/- 0.35degrees C for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/microL), and quantifiable (R 2 = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.
Animals ; Cattle ; Cattle Diseases/epidemiology/microbiology ; Escherichia coli Infections/epidemiology/microbiology/*veterinary ; Feces/microbiology ; Multiplex Polymerase Chain Reaction/veterinary ; Nucleic Acid Amplification Techniques/*veterinary ; Shiga Toxin 1/*genetics/isolation & purification ; Shiga Toxin 2/*genetics/isolation & purification ; Shiga-Toxigenic Escherichia coli/*genetics/isolation & purification

Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from 10(-1) to 10(-5) ng/microl for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of 63degrees C by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha (EF1alpha) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis.
Animals ; Dog Diseases/*diagnosis/parasitology ; Dogs ; Feces/parasitology ; Giardia lamblia/genetics/*isolation & purification ; Giardiasis/diagnosis/parasitology/*veterinary ; Humans ; Molecular Diagnostic Techniques/*methods ; Molecular Sequence Data ; Nucleic Acid Amplification Techniques/*methods ; Pets ; Sensitivity and Specificity ; Sequence Analysis, DNA ; Temperature ; Time Factors ; Veterinary Medicine/*methods

A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods.
Animals ; Azure Stains ; Biological Assay ; Brain/parasitology ; DNA, Protozoan/*blood/genetics ; Lung/parasitology ; Mice ; Nucleic Acid Amplification Techniques/*veterinary ; Parasitemia ; Real-Time Polymerase Chain Reaction/veterinary ; Sensitivity and Specificity ; Swine ; Swine Diseases/*diagnosis/parasitology ; Toxoplasma/genetics/*isolation & purification ; Toxoplasmosis, Animal/*diagnosis/parasitology

Epidemiological situation of taeniasis in Mongolia was assessed based on mitochondrial DNA identification of the parasite species. Multiplex PCR was used on a total of 194 proglottid specimens of Taenia species and copro-PCR and loop-mediated isothermal amplification (LAMP) assays were utilized for detection of copro-DNA of 37 fecal samples from taeniasis patients submitted to the Mongolian National Center for Communicable Diseases (NCCD) from 2002 to 2012. In addition, 4 out of 44 calcified cysts in beef kept in formalin since 2003 were evaluated for histopathological confirmation of cattle cysticercosis. All proglottid specimens and stool samples were confirmed to be Taenia saginata by multiplex PCR and by copro-PCR and LAMP, respectively. Cysts collected from cattle were morphologically confirmed to be metacestodes of Taenia species. T. saginata taeniasis was identified from almost all ages from a 2-year-old boy up to a 88-year-old woman and most prominently in 15-29 age group (37%, 74/198) followed by 30-44 age group (34.8%, 69/198 ) from 15 of Mongolia's 21 provinces, while cattle cysticerci were found from 12 provinces. The highest proportion of taeniasis patients was in Ulaanbaatar, the capital of Mongolia.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Animals ; Cattle/parasitology ; Child ; Child, Preschool ; Cysticercosis/*epidemiology/parasitology ; DNA, Helminth/*genetics ; DNA, Mitochondrial/*genetics ; Feces/parasitology ; Female ; Geography ; Humans ; Male ; Meat/parasitology ; Middle Aged ; Mitochondria/genetics ; Mongolia/epidemiology ; Neglected Diseases/epidemiology ; Nucleic Acid Amplification Techniques/veterinary ; Questionnaires ; Taenia saginata/*genetics ; Taenia solium/genetics ; Taeniasis/*epidemiology/parasitology ; Young Adult

A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.
Animals ; Colorimetry ; methods ; DNA Primers ; genetics ; Electrophoresis, Agar Gel ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; Influenza A Virus, H3N2 Subtype ; genetics ; Influenza, Human ; diagnosis ; virology ; Naphthalenesulfonates ; chemistry ; Nucleic Acid Amplification Techniques ; methods ; Orthomyxoviridae Infections ; diagnosis ; veterinary ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Swine ; Swine Diseases ; diagnosis ; virology ; Temperature