Loop Primers Research Articles

Abstract 3966: Platelets possess prostate cancer specific miRNAs: Implications for a novel source of biomarkers

Abstract Introduction and Objective: Our previous studies demonstrated that some circulation-bound tumor-derived factors are taken up and concentrated by circulating platelets, and are released upon platelet activation. MicroRNAs (miRNAs) are small, non-coding nucleic acid molecules involved in regulation of gene expression that have potential as biomarkers in prostate cancer. Prior descriptions of miRNA presence in platelets of healthy individuals indicates that uptake of tumor-derived miRNAs may be reflected by changes in platelet miRNA signature. To date, in the prostate cancer setting, the role of platelets as a source of miRNA-based biomarkers has not been investigated. The aim of this study was to ascertain the presence of prostate cancer-associated miRNAs in platelets from patients with aggressive disease and explore the potential use of platelets as a source of miRNA-based biomarkers. Methods Patients with aggressive prostate cancer (n = 12) were identified at a large, tertiary care medical center, informed of the study and consented to provide whole blood samples. Platelets were isolated from venous blood using citrate containing tubes and purified by serial centrifugations and washings. Platelet purification was confirmed at >99.9% by flow cytometry and platelet specific antibodies. Total RNA was extracted from platelets of men with aggressive prostate cancer (n = 2), low risk disease (n = 1) and age matched (50+) controls (n = 2). By performing a Real Time-quantitative PCR (RT-qPCR) profiling using the Human Panel I (Exiqon) (which encompasses 373 microRNAs representing the best-established and well-annotated human miRNAs) we identified several miRNAs as potential candidates of the aggressive disease. TaqMan miRNA Reverse Transcription kit and miRNA-specific stem loop primers were then used to validate the presence of miRNA candidates identified by the array in platelets from patients with aggressive disease (n = 10). All RT-qPCR reactions were performed in triplicates using TaqMan 2x Universal PCR Master Mix. Results Eleven of 12 patients in our initial study had castrate-resistant prostate cancer (the remaining one was characterized as having low risk prostate cancer). Patient ages ranged from 57 to 82. Upregulated expression of the following prostate cancer-specific miRNA molecules in platelets were identified: mir-141 (n = 7/12 patients), mir-134 (6/12), mir-181c (7/12), mir-193 (3/12),and mir-132 (7/12). Conclusions Our findings indicate that platelets may potentially represent a novel source of biomarkers in prostate cancer. Citation Format: Brady Miller, Mackenzie Adams, Harene Venghatakrishnan, Maria Giraldez, Muneesh Tewari, Ganesh Palapattu, Alexander Zaslavsky. Platelets possess prostate cancer specific miRNAs: Implications for a novel source of biomarkers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3966. doi:10.1158/1538-7445.AM2015-3966

Real-time sequence-validated loop-mediated isothermal amplification assays for detection of Middle East respiratory syndrome coronavirus (MERS-CoV).

The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.

Rapid detection of blaNDM, blaKPC, blaIMP, and blaVIM carbapenemase genes in bacteria by loop-mediated isothermal amplification.

A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for rapid detection of blaKPC, blaNDM, blaIMP, and blaVIM carbapenemase genes. Six oligonucleotides, including outer, inner, and loop primers, were designed for eight distinct regions in each target gene. Two qualitative criteria were used to evaluate LAMP reactions: visual inspection of color change and real-time detection of fluorescence change. The lower detection limit was 10 colony forming units (CFU) per reaction for real-time detection and 100 CFU per reaction for visual inspection for each gene. Two hundred twenty-two carbapenem-resistant clinical isolates (including 100 Pseudomonas aeruginosa, 100 Acinetobacter sp., and 22 Enterobacteriaceae) were tested by LAMP assay. At the same time, these isolates were confirmed by conventional polymerase chain reaction (PCR) and sequencing analysis. In these clinical isolates, the results of 11 strains with blaNDM, 11 strains with blaKPC, 11 strains with blaVIM, and 2 strains with blaIMP obtained using LAMP assays were concordant with conventional PCR. The LAMP method reported here may be a useful and powerful tool for rapid detection of blaNDM, blaKPC, blaIMP, and blaVIM carbapenemase genes in bacteria.

Bovine embryo sex determination by multiplex loop-mediated isothermal amplification

In cattle, the ability to determine the sex of embryos before embryo transfer is beneficial for increasing the number of animals with the desired sex. This study therefore developed a new modification of loop-mediated isothermal amplification in a multiplex format (multiplex LAMP) for highly efficient bovine embryo sexing. Two chromosomal regions, one specific for males (Y chromosome, S4 region) and the other common to both males and females (1.715 satellite DNA), were amplified in the same reaction tube. Each target was amplified by specifically designed inner primers, outer primers, and loop primers, where one of the S4 loop primers was labeled with the fluorescent dye 6-carboxyl-X-rhodamine (emitting a red color), whereas both satellite loop primers were labeled with the fluorescent dye fluorescein isothiocyanate (emitting a green color). After amplification at 63 °C for 1 hour, the amplified products were precipitated by a small volume of cationic polymer predispensed inside the reaction tube cap. Green precipitate indicated the presence of only control DNA without the Y chromosome, whereas orange precipitate indicated the presence of both target DNAs, enabling interpretation as female and male, respectively. Accuracy of the multiplex LAMP assay was evaluated using 46 bovine embryos with known sex (25 male and 21 female) generated by somatic cell nuclear transfer and confirmed by multiplex polymerase chain reaction. The multiplex LAMP showed 100% accuracy in identifying the actual sex of the embryos and provides a fast, simple, and cost-effective tool for bovine embryo sexing.

Abstract 5199: Distinct expression pattern of microRNAs bioinformatically predicted to target SATB1 in human breast cancer

Abstract Recently, genome organizer protein special AT-rich sequence-binding protein 1(SATB1) has been reported to promote a metastatic phenotype and correlate with poor prognosis in breast cancer. The mechanism of SATB1 elevation in breast cancer remains elusive. This study was to investigate whether microRNAs (miRNAs) play a role in modulating SATB1 expression in ductual breast cancer by using archival formalin-fixed and paraffin-embedded (FFPE) clinical samples. Firstly, four canonical target prediction databases, including TargetScan, PicTar, miRanda and miRDB, were utilized to predict the interaction between miRNA and the 3′-UTR of SATB1 mRNA. The results of the prediction software programs were then integrated by displaying Venn diagram. Secondly, the Qiagen miRNeasy FFPE kit was used to isolate total RNA from 93 specimens with primary invasive ductual breast cancer. Expression of candidate miRNA transcriptional levels was measured using TaqMan MicroRNA Assays with miRNA-specific stem loop primers by relative real-time quantitative RT-PCR. The relative levels of miRNA expression were calculated from the relevant signals by normalization with the signal for RNU6B miRNA expression in each sample. Meanwhile, SATB1 protein expression levels were assessed by immunohistochemistry(IHC). Finally, correlations between the expression levels of these miRNAs and SATB1 were analyzed. Overall 22 miRNAs, 24 miRNAs, 76 miRNAs and 75 miRNAs were found by TargetScan, PicTar, miRanda and miRDB, to target SATB1 respectively, whereas only miR-7, miR-21, miR-23a, miR-23b, miR-34a and miR-155 were found by all four programs to be predicted to regulate SATB1. Quantitative RT-PCR detection analysis showed that expression levels of miR-34a were much higher in SATB1-negative than in SATB1-positive tumors (p<0.001). whereas lower miR-7 expression showed a strong trend toward an association with elevated SATB1 expression (P = 0.056). We then extended the analysis of miR-34a expression in breast cancer tissues to include all scores for SATB1 protein expression, assessed semiquantitatively by IHC. miR-34a expression levels were gradually decreased as SATB1 protein scores increased. Moreove, our quantitative RT-PCR results showed no differences in miR-21, miR-23a, miR-23b, and miR-155 expression between SATB1-negative and SATB1-positive tumors. We thus conclude that SATB1 maybe a novel target of miR-34a in human breast cancer. Downregulation of miR-34a promotes human breast cancer to progress through reduced repression of SATB1. Restoration of miR-34a may prove therapeutic in breast cancer patients in which aberrant SATB1 expression plays a role. Note: This abstract was not presented at the meeting. Citation Format: Weiming Duan, Zixing Chen, Li Yao, Min Tao, Kai Chen, Wei Liu, Jiannong Cen. Distinct expression pattern of microRNAs bioinformatically predicted to target SATB1 in human breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2014-5199

Rapid and sensitive detection of type II porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was combined with a vertical flow (VF) nucleic acid detection strip to develop a universal assay for the detection of type II porcine reproductive and respiratory syndrome virus (PRRSV). The loop primers were labeled separately with biotin and fluorescein isothiocyanate (FITC) in this assay. Using optimized parameters, the whole reaction could be completed in <50min in a completely enclosed environment. The detection limit of this assay was found to be 1pg RNA, 30 tissue culture infective dose 50 (TCID50) virus, or 230 copies of recombinant plasmid DNA, which is relatively higher than that of RT-LAMP analyzed by agarose gel, RT-LAMP visualized by calcein, and the conventional RT-polymerase chain reaction (PCR). No false-positive results were obtained in the specificity assay. The efficiency of the RT-LAMP method was tested by analyzing 43 clinical samples, and the results were compared with those obtained by RT-PCR analysis, with the respective positive rates of 32.56% and 27.91%. This result confirmed that the method described is a rapid, accurate, and sensitive method for universal type II PRRSV detection. Also, this method can be used for the rapid detection of type II PRRSV during the early phase of an outbreak, especially for rapid veterinary diagnosis on the spot and in rural areas.

Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.

Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings.

Development and Application of a Novel Nucleic Amplification Kit on Detection of Several Pathogens

Escherichia coli O157, Psuedomonas aeruginosa, Salmonella, Vibrio parahaemolyticus and Listeria are important pathogens for human. With increased awareness in public health, development of a rapid, sensitive, cost-effective and easy-operating bacteriological detection is of the utmost importance and urgent necessity. In this study, we developed and applied a simple amplification kit based on loop-mediated isothermal amplification (LAMP) methods for rapid detection of various pathogens including Escherichia coli O157, Psuedomonas aeruginosa, Salmonella, Vibrio parahaemolyticus and Listeria, as well as related virulence. Nine targets, including rfbE (E. coli-specific), stx1 (coding for Shiga toxin 1), stx2 (coding for Shiga toxin 2), oprI (P. aeruginosa–specific), invA (Salmonella-specific), hlyA (Listeria-specific), tlh (coding for thermolable haemolysin), tdh (coding for thermostable direct haemolysin) and trh (coding for TDH-related haemolysin), were selected for identification for Escherichia coli O157, Psuedomonas aeruginosa, Salmonella, Vibrio parahaemolyticus and Listeria, as well as related virulence.. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on the targets. Three solutions labeled A, B and C was included in the kit. The experiment were carried out in a total of 25 μl reaction mixture: solution A containing 1.6 μM (each) of the primers FIP and BIP, 0.2 μM (each) of the primers F3 and B3, 0.8 μM (each) of primers LF and LB; solution B containing 1.6 mM of deoxynucleoside triphosphates, 6 mM MgSO4, 1 M betain (Sigma, St. Louis, MO, USA), 1 X thermopol buffer (New England Biolabs, Ipswich, MA, USA); solution C containing BST polymerase. Twenty-two reference strains, including various species of gram-negative and-positive isolates, were included in this study to evaluate and optimize LAMP assays. Application of the optimized LAMP assays was performed on a total of 200 strains (with 40 strains for each species of the pahtogens). The optimal reaction condition was found to be 65°C for 45 min. Application of the kit assays were performed on various types of pathogens, the sensitivity for the 9 targets was found to be 100%; with a 100% specificity and positive predictive value (PPV) for all the 9 targets targets. In conclusion, the isothermal amplification kits were demonstrated to be useful and powerful tools for rapid differentiation of various pathogens (including Escherichia coli O157, Psuedomonas aeruginosa, Salmonella, Vibrio parahaemolyticus and Listeria), and undoubtedly, the rapidness, easiness and cost-effectiveness of LAMP assay will aid in the broad application of bacteriological detection of common pathogens.

Development and Application of a Novel Nucleic Amplification Kit on Rapid Detection of Various Types of Staphylococci Strains

A loop-mediated isothermal amplification (LAMP) method for rapid detection of various staphylococci strains and associated antibiotic resistance determinant had been developed and evaluated in this study. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on four targets: 16Sr RNA, femA, mecA, and orfX. Twenty-seven reference strains, including various species of gram-negative and-positive isolates, were included in this study to evaluate and optimize LAMP assays. The optimal reaction condition was found to be 65°C for 45 min, with detection limits at 100 fg DNA/tube and 10 CFU/reaction for 16S rRNA, 100 fg DNA/tube and 10 CFU/reaction for femA, 1 pg DNA/tube and 100 CFU/reaction for mecA, 10 DNA/tube and 10 CFU/reaction for orfX, respectively. Application of LAMP assays were performed on 166 various types of staphylococci isolates, the detection rate of LAMP assays for the 16Sr RNA, femA, mecA, and orfX was 100% (166/166), 98.5% (64/65), 94.3% (66/70), and 98.6% (69/70) and the negative predictive value (NPV) was 100%, 98.1%, 92.3%, and 92.7% respectively; with a 100% positive predictive value (PPV) for all three targets. In conclusion, LAMP assays were demonstrated to be useful and powerful tools for rapid detection of various staphylococci strains.

OP0256 Changes of Microrna Expression in Lymph Node Stromal Cells of Rheumatoid Arthritis Patients

BackgroundLymph node stromal cells (LNSC) build the scaffold that enables migration and interaction of lymphocytes in the lymph node. More recently, it has been shown that during inflammation LNSC play...

Loop-mediated isothermal amplification of DNA (LAMP): a new diagnostic tool lights the world of diagnosis of animal and human pathogens: a review.

Diagnosis is an important part in case of animal husbandry as treatment of a disease depends on it. Advancement in molecular biology has generated various sophisticated tools like Polymerase Chain Reaction (PCR), its versions along with pen-side diagnostic techniques. Every diagnostic test however has both advantages and disadvantages; PCR is not an exception to this statement. To ease the odds faced by PCR several non-PCR techniques which can amplify DNA at a constant temperature has become the need of hour, thus generating a variety of isothermal amplification techniques including Nucleic Acid Sequence-Based Amplification (NASBA) along with Self-Sustained Sequence Replication (3SR) and Strand Displacement Amplification (SDA) and Loop mediated isothermal amplification (LAMP) test. LAMP stands out to be a good and effective diagnostic test for empowering in developing countries as it does not require sophisticated equipments and skilled personnel and proves to be cost-effective. Performance of LAMP mainly relies on crafting of six primers (including 2 loop primers) ultimately accelerating the reaction. LAMP amplifies DNA in the process pyrophosphates are formed causing turbidity that facilitates visualisation in a more effective way than PCR. The Bst and Bsm polymerase are the required enzymes for LAMP that does not possess 5'-3' exonuclease activity. Results can be visualized by adding DNA binding dye, SYBR green. LAMP is more stable than PCR and real-time PCR. Non-involvement of template DNA preparation and ability to generate 10(9) copies of DNA are added benefits that make it more effective than NASBA or 3SR and SDA. Thus, it fetches researcher's interest in developing various versions of LAMP viz., its combination with lateral flow assay or micro LAMP and more recently lyophilized and electric (e) LAMP. Availability of ready to use LAMP kits has helped diagnosis of almost all pathogens. LAMP associated technologies however needs to be developed as a part of LAMP platform rather than developing them as separate entities. This review deals with all these salient features of this newly developed tool that has enlightened the world of diagnosis.

Protocol: a beginner's guide to the analysis of RNA-directed DNA methylation in plants.

BackgroundDNA methylation is a conserved epigenetic mark that controls genome stability, development and environmental responses in many eukaryotes. DNA methylation can be guided by non-coding RNAs that include small interfering RNAs and scaffold RNAs. Although measurement of DNA methylation and regulatory non-coding RNAs is desirable for many biologists who are interested in exploring epigenetic regulation in their areas, conventional methods have limitations and are technically challenging. For instance, traditional siRNA detection through RNA hybridization requires relatively large amount of small RNAs and involves radioactive isotopes. An alternative approach is RT-qPCR that employs stem loop primers during reverse transcription; however, it requires a prerequisite that the exact sequences of siRNAs should be known.ResultsBy using the model organism Arabidopsis thaliana, we developed an easy-to-follow, integrative procedure for time-efficient, quantitative measurement of DNA methylation, small interfering RNAs, and scaffold RNAs. Starting with simplified nucleic acid manipulation, we examined DNA methylation levels by using Chop PCR (methylation-sensitive enzyme digestion followed by PCR), which allowed for fast screening for DNA methylation mutants without the need of transgenic reporters. We deployed a simple bioinformatics method for mining published small RNA databases, in order to obtain the nucleotide (nt) sequences of individual 24nt siRNAs within the regions of interest. The protocol of commercial TaqMan Small RNA Assay was subsequently optimized for reliable quantitative detection of individual siRNAs. We used nested qPCR to quantify scaffold RNAs that are of low abundance and without Poly-A tails. In addition, nuclei fraction enables separation of chromatin-associated scaffold RNAs from their cognate non-scaffold transcripts that have been released from chromatin.ConclusionsWe have developed a procedure for quantitative investigations on nucleic acids that are core components of RNA-directed DNA methylation. Our results not only demonstrated the efficacy of this procedure, but also provide lists of methylation-sensitive restriction enzymes, novel DNA methylation marker loci, and related siRNA sequences, all of which can be valuable for future epigenetic studies. Importantly, step-by-step protocols are provided in details such that the approaches can be easily followed by biologists with little experience in epigenetics.

Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25.

The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg−1 GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25.

Comparison of Loop-Mediated Isothermal Amplification and PCR for the Detection and Differentiation of Marek's Disease Virus Serotypes 1, 2, and 3

The previously conducted study on loop-mediated isothermal amplification (LAMP) has shown its usefulness for the detection of Marek's disease virus (MDV) virulent field strains. The current study improves the previously designed LAMP method with an additional pair of loop primers, which accelerates the reaction, and describes two other LAMP procedures for the specific detection of FC126 strain of turkey herpesvirus and nonpathogenic SB-1 strain. The developed LAMP procedures were also confirmed and compared with PCR. Each LAMP reaction used three pairs of specific primers designed to target the nucleotide sequence of the very virulent MDV strain, the SB-1 strain of MDV-2, and turkey herpesvirus, respectively. All LAMP reactions were flexible and provided reliable results at a wide range of incubation temperatures from 54.0 to 62.3 C in 15 to 90 min. LAMP does not need any thermocyclers, because all assays were conducted in a water bath. The green fluorescence signal was recorded under ultraviolet illumination in LAMP samples containing virulent MDV and turkey herpesvirus where SYBR Green was added to the reaction mixture, whereas the SB-1-positive samples presented orange illumination after GelRed staining solution. The sensitivity of the three LAMP reactions ranged from 2 log10 plaque-forming units (PFU)/ml of the virulent MDV HPRS-16 strain and turkey herpesvirus (HVT) to 3 log10 PFU/ml of the SB-1 nonpathogenic strain. The sensitivity of the compared PCR was lower by 1-2 log10 PFU/ml. The conducted studies have shown that developed LAMP methods may be used instead of PCR for the detection and differentiation of virulent and nonpathogenic MDV strains used in prophylaxis against MD. LAMP may be conducted without access to thermocyclers.

A loop-mediated isothermal amplification method for the detection of members of the genus Ranavirus

A loop-mediated isothermal amplification (LAMP) method was developed for detection of members of the genus Ranavirus. The optimum reaction mixture contained 2.5 μL of each inner primer, RV-FIP (20pmol/μL) and RV-BIP (20pmol/μL), 0.5 μL of each outer primer, RV-F3 (10pmol/μL) and RV-B3 (10pmol/μL), 1.25 μL of each loop primer, RV-LF (20pmol/μL) and RV-LB (20pmol/μL), 3.5 μL dNTP mix (10mM each), 8 μL MgSO4 (25mM), 1 μL of Bst DNA polymerase (8 U/mL, large fragment; New England Biolabs Inc., Beverly, MA, USA), 2.5 μL 10×supplied buffer, and 1 μL of template DNA in a final volume of 25 μL. The optimum reaction conditions were 63 °C for 60min. This LAMP method could detect Andrias davidianus iridovirus (ADIV), soft-shelled turtle iridovirus (STIV), and epizootic hematopoietic necrosis virus (EHNV), all of which belong to the genus Ranavirus, but it could not detect other viruses such as koi herpes virus (KHV), channel catfish virus (CCV), infectious spleen and kidney necrosis virus (ISKNV) and white spot syndrome virus (WSSV). The detection limit of the LAMP method was 100 copies of STIV DNA segment, and the sensitivity was 10 times higher than that of the polymerase chain reaction (PCR) assay. The results could be estimated visually by eye when calcein was added.

Detection of capripoxvirus DNA using a novel loop-mediated isothermal amplification assay

BackgroundSheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants. They are double stranded DNA viruses of the genus Capripoxvirus, (subfamily Chordopoxvirinae) within the family Poxviridae. The aim of this study was to develop a Loop-mediated isothermal AMPlification (LAMP) assay for the detection of Capripoxvirus (CaPV) DNA.ResultsA single LAMP assay targeting a conserved region of the CaPV P32 gene was selected from 3 pilot LAMP assays and optimised by adding loop primers to accelerate the reaction time. This LAMP assay successfully detected DNA prepared from representative CaPV isolates (SPPV, GTPV and LSDV), and did not cross-react with DNA extracted from other mammalian poxviruses. The analytical sensitivity of the LAMP assay was determined to be at least 163 DNA copies/μl which is equivalent to the performance reported for diagnostic real-time PCR currently used for the detection of CaPV. LAMP reactions were monitored with an intercalating dye using a real-time PCR machine, or by agarose-gel electrophoresis. Furthermore, dual labelled LAMP products (generated using internal LAMP primers that were conjugated with either biotin or fluorescein) could be readily visualised using a lateral-flow device.ConclusionsThis study provides a simple and rapid approach to detect CaPV DNA that may have utility for use in the field, or in non-specialised laboratories where expensive equipment is not available.

Abstract 1166: Upregulation of microRNAs in colorectal tumors and association with disease stage.

Abstract Introduction MicroRNAs (miRNAs) are small, noncoding RNAs that function as regulators of many critical cellular processes. Increased expression of specific microRNAs in tumor tissue may be indicative of a patient's prognosis. The purpose of this study was to assess miRNA upregulation in fresh frozen colorectal cancer (CRC) tumors compared to paired normal colorectal tissue in a prospective cohort of CRC patients. Methods Tumor and normal tissue were collected at the time of surgery from 40 stage I-IV CRC patients participating in the ColoCare Study in Seattle, WA. The Qiagen miRNeasy kit was used to isolate miRNAs for analysis. Expression of candidate miRNAs (e.g., miR-141, miR-31, miR-29a, mir-92a, miR-203) was measured using Taqman qRT-PCR with miRNA-specific stem loop primers. For each miRNA, copy number was determined via standard curves and normalized to miR-16 expression for each sample. Expression in tumor was compared to expression in paired normal tissue for each participant. In addition, we profiled two stage III CRC tumor-normal pairs using the miCURY LNA human panel I qRT-PCR array from Exiqon. Relative expression from the Exiqon panel was calculated using GenEx software and endogenous control assays. The Wilcoxon two sample test was used to compare normalized miRNA upregulation between groups. Results Expression of miR-31 was greater than 10-fold higher in tumor compared to normal tissue for 13 of 38 tumor-normal pairs with Taqman data available. Overexpression of miR-31 in tumor tissue was significantly correlated with stage III-IV CRC when compared to stage I-II CRC (p&amp;lt;0.01). The Taqman and miCURY platforms both showed miR-31 upregulation in the two stage III CRC tumors, but the extent of upregulation varied between platforms. Conclusion In this study, upregulation of miR-31 in CRC tumor tissue was associated with more advanced disease stage in CRC patients. Future studies will investigate additional miRNAs, and associations with clinicopathological features, patient outcomes, and circulating miRNA levels. Citation Format: Karen W. Makar, Michelle A. Wurscher, Nina Habermann, William M. Grady, Cornelia M. Ulrich. Upregulation of microRNAs in colorectal tumors and association with disease stage. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1166. doi:10.1158/1538-7445.AM2013-1166

Loop-Mediated Isothermal Amplification as a Simple Molecular Method for the Detection of Derzsy’s Disease Virus

Abstract The objective of the study was to develop a simple and rapid molecular method for the detection of GPV. Twenty seven goose parvovirus (GPV) isolates collected from geese flocks in Poland were examined. Three pairs of specific primers: two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (FL and BL) were used to accelerate the reaction. The optimum temperature and time of the reaction were 60°C and 30 min. The sensitivity of the method was 10-times higher than PCR. The method proved to be a sensitive, rapid, and specific assay for detecting GPV.

An improved method for detection of Edwardsiella tarda by loop-mediated isothermal amplification by targeting the EsrB gene.

Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses. An improved method for quick and accurate detection of E. tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene (EsrB — LAMP). In this method, the Mg2+ concentration, reaction temperature, and reaction time were optimized to 8 mmol/L, 61°C, and 40 min, respectively. The detection limit with the EsrB gene was as low as 10 copies, which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR). The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene ( hemolysin -LAMP) for detection of E. tarda. The EsrB -LAMP was also highly specific to E. tarda and had no cross-reaction with 13 other strains of bacteria. The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture. Taken together, the improved EsrB-LAMP diagnostic protocol has the potential for detection of E. tarda from indoor and outdoor samples.

Loop-mediated isothermal amplification for the detection of goose circovirus

BackgroundGoose circovirus (GCV) presents an immunosuppressive problem in production of geese. The infection’s clinical symptoms include growth retardation or feathering disorders but the infection process may remain non-symptomatic what makes the infected birds more susceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection is made by histopathological examination, dot blot hybridization, polymerase chain reaction (PCR) and real-time PCR. However these techniques require application of thermocyclers and qualified staff which may be cost-consuming for some diagnostic units. The aim of this study was to develop loop-mediated isothermal amplification assay (LAMP) as a simple method of GCV detection.ResultsThe presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus (GCV) as soon in 30 min time. The method used three sets of primers: two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers (FL and BL) to accelerate the reaction. The optimum reaction temperature and the time were 61°C for 30 min, respectively. The results were analysed using SYBR Green dye and GelRedTM solutions. Thirty-eight isolates of GCV collected from geese flocks in Poland were examined. For comparison, real-time polymerase chain reaction with F3 and B3 primers and SYBR Green dye was conducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specific assay and alternative for PCR-based methods.ConclusionsThe developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV.