Genome-wide association analyses identified novel susceptibility loci for pulmonary embolism among Han Chinese population.

BACKGROUND: A large proportion of pulmonary embolism (PE) heritability remains unexplained, particularly among the East Asian (EAS) population. Our study aims to expand the genetic architecture of PE and reveal more genetic determinants in Han Chinese. METHODS: We conducted the first genome-wide association study (GWAS) of PE in Han Chinese, then performed the GWAS meta-analysis based on the discovery and replication stages. To validate the effect of the risk allele, qPCR and Western blotting experiments were used to investigate possible changes in gene expression. Mendelian randomization (MR) analysis was employed to implicate pathogenic mechanisms, and a polygenic risk score (PRS) for PE risk prediction was generated. RESULTS: After meta-analysis of the discovery dataset (622 cases, 8853 controls) and replication dataset (646 cases, 8810 controls), GWAS identified 3 independent loci associated with PE, including the reported loci FGG rs2066865 (p-value = 3.81 × 10-14), ABO rs582094 (p-value = 1.16 × 10-10) and newly reported locus FABP2 rs1799883 (p-value = 7.59 × 10-17). Previously reported 10 variants were successfully replicated in our cohort. Functional experiments confirmed that FABP2-A163G(rs1799883) promoted the transcription and protein expression of FABP2. Meanwhile, MR analysis revealed that high LDL-C and TC levels were associated with an increased risk of PE. Individuals with the top 10% of PRS had over a fivefold increased risk for PE compared to the general population. CONCLUSIONS: We identified FABP2, related to the transport of long-chain fatty acids, contributing to the risk of PE and provided more evidence for the essential role of metabolic pathways in PE development.

Programmable Biosensors Based on RNA-Guided CRISPR/Cas Endonuclease.

Highly infectious illnesses caused by pathogens constitute severe threats to public health and lead to global economic loss. The use of robust and programmable clustered regularly interspaced short palindromic repeat and CRISPR-associated protein (CRISPR-Cas) systems, repurposed from genome-engineering applications has markedly improved traditional nucleic acid detection for precise identification, independently enabling rapid diagnostics of multiplex biomarker with genetic and mutation related to tumors, and microbial pathogens. In this review, we delineate the utility of the current CRISPR-Cas enzyme as biosensors by which these effector toolkits achieve recognition, signaling amplification, and finally, accurate detection. Additionally, we discuss the details of the dominance and hurdles related to expanding this revolutionary technology into an effective and convenient contraption crucial for improving the rational redesign to CRISPR/Cas biosensing. Overall, this review provides an insight into the current status of rapid and POC diagnostic systems by CRISPR/Cas tools.

Electrically injected GaN-on-Si blue microdisk laser diodes.

III-nitride blue microdisk laser diodes are highly desirable in emerging applications, such as augmented reality, virtual reality, and visible light communication. However, the electrically pumped blue microdisk lasers have been lagging for decades owing to weak optical confinement and large internal absorption loss. In this study, the waveguide layers and cladding layers were carefully engineered to enhance the optical confinement and reduce internal absorption loss. Therefore, the first electrically injected blue microdisk laser diodes grown on Si substrates have been successfully fabricated, and exhibited a resistor-capacitance-limited bandwidth of 24.1 GHz, showing highly promising applications in high-speed and large-modulation-bandwidth visible light communication.

Tuberous sclerosis complex with skin lesions as the initial presentation: A case report and multidisciplinary discussion. / 1例以皮肤损害为首发症状的结节性硬化症并多学科讨论.

We reported a case of tuberous sclerosis complex with facial angiofibroma as the initial presentation and conducted a multidisciplinary discussion. The patient, a young female, was admitted to the Department of Dermatology for cosmetic purpose. After the examination, she was found to have multiple system involvement, including a large renal angiomyolipoma pressing on the liver. She never had any subjective symptom. After consultation by the multidisciplinary team of tuberous sclerosis complex, the patient was treated with everolimus orally and followed up regularly. It is suggested that dermatologists should pay attention to the systemic involvement of patients with tuberous sclerosis complex. Early intervention can prolong the life of patients and improve their life quality. Multidisciplinary collaboration for lifelong disease management is the key to enhance the diagnosis and treatment for tuberous sclerosis complex and enhance the prognosis of patients.

Activation of buried p-GaN through nanopipes in large-size GaN-based tunnel junction LEDs.

In GaN-based light-emitting diodes (LEDs), tunnel junctions offer a way of replacing the highly resistive p-type GaN (p-GaN) ohmic contact with a low-resistance n-GaN ohmic contact. However, the p-GaN would be re-passivated by hydrogen atoms during the subsequent growth of n-GaN in a metal-organic chemical vapor deposition (MOCVD) chamber. The n-GaN layer, acting as a hydrogen diffusion barrier, hinders the thermal activation of the underlying p-GaN. Here, we report a method to thermally activate the buried p-GaN in tunnel junction LED (TJ-LED) through vertically aligned nanopipe arrays across the top n-GaN layer, which provides a hydrogen outgassing passage. The fabrication of nanopipes is realized via inductive coupled plasma etching using a mask prepared by self-assembled nanosphere arrays. As a result, we attain large-size TJ-LED chips, exhibiting nearly equivalent p-GaN activation and superior light extraction compared to conventional LEDs. Specifically, the light extraction efficiency is boosted by 44% relative to conventional LEDs at an injection current density of 100 A cm-2.

Effects of STIP1 and GLCCI1 polymorphisms on the risk of childhood asthma and inhaled corticosteroid response in Chinese asthmatic children.

BACKGROUND: Asthma is a common chronic lung disease in children. We aimed to determine the associations between stress-induced phosphoprotein 1 (STIP1) and glucocorticoid-induced transcript 1 (GLCCI1) polymorphisms and susceptibility of childhood asthma and inhaled corticosteroid (ICS) response in children. METHODS: A total of 263 Chinese Han asthmatic children were recruited from the Xiangya Hospital, Central South University. Pulmonary function tests were performed before the treatment and 3 months after the treatment. One hundred fifty non-asthmatic children were recruited. Each participant's DNA was extracted from the peripheral blood and Method of MassARRAY was used to genotype the single-nucleotide polymorphisms (SNPs). RESULTS: STIP1 rs2236647 wild-type homozygote (CC) was associated with increased asthma risk of children (OR = 1.858, 95% CI:1.205-2.864), but not associated with the ICS response. GLCCI1 rs37969, rs37972 and rs37973 polymorphisms were not associated with the risk of childhood asthma. However, rs37969 mutant genotypes (TT/GT) were significantly associated with less improvement in PD20 (p = 0.028). We also found significant associations between rs37969, rs37972 and rs37973 mutant genotypes and less improvement in maximal midexpiratory flow (MMEF) after ICS treatment for 3 months (p = 0.036, p = 0.010 and p = 0.003, respectively). CONCLUSIONS: STIP1 rs2236647 was associated with asthma risk of children and GLCCI1 rs37969 mutant genotypes were associated with less improvement in airway hyper-responsiveness. GLCCI1 rs37969, rs37972 and rs37973 polymorphisms might be associated with pulmonary function in childhood asthma patients after ICS treatment.

[Cyanosis for more than 4 years in a girl aged 4 years and 3 months].

A girl, aged 4 years and 3 months, presented with cyanosis of the lips shortly after birth. She then experienced shortness of breath after activity 1 year ago and acrocyanosis 3 months ago, with obvious acropachy and toe deformity. Laboratory examinations revealed an increase in hemoglobin (178 g/L) and a reduction in arterial partial pressure of oxygen (37.7 mm Hg). Plain and contrast-enhanced CT scans of the lungs showed a large area of dense shadow and multiple nodules with clear boundaries in the right lower lung, as well as thickening of the arteries and dilatation of the veins in the right lower lung. Magnetic resonance angiography of the pulmonary artery showed large arteriovenous malformation in the lung. The child was diagnosed with congenital pulmonary arteriovenous fistula and was given interventional embolization of the pulmonary arterial fistula. The child was followed up at 3 months after surgery. The symptoms of shortness of breath and cyanosis disappeared, and activity tolerance, heart rate, hemoglobin, red blood cell count, and transcutaneous oxygen saturation all returned to normal.

Expression of recombinant tachyplesin I in Pichia pastoris.

The development of antibiotic-resistant bacteria has become a major public health problem, prompting the search for alternative solutions. Tachyplesin I (TP-I) is an antimicrobial peptide, which exhibits potent and broad-spectrum activities against bacteria, fungi, viruses, and tumor cells. However, limited amounts of TP-I produced in horseshoe crab restrict its large-scale use. In order to solve this problem, a eukaryotic expression system of Pichia pastoris with high TP-I expression was constructed by gene engineering. To achieve high expression of TP-I, 74 amino acid-long peptide (4TP-1) was designed containing 4 copies of TP-I, and specific cleavage sites for pancreatic elastase (-Ala↓ or -Gly↓) and carboxypeptidase A (cleaves C terminal amino acid); these cleavage sites for enzymes were located between the four copies of TP-I. The gene sequence for the designed peptide was synthesized taking into consideration codon preferences for P. pastoris, and cloned into the highly efficient expression vector pGAPZα B. Host Pichia pastoris strain GS115 cells were transfected by the constructed expression vector pGAPZα B-4tp-I by electroporation. Tricine-SDS-PAGE electrophoresis was carried out to detect the expression of target peptides in the fermentation medium. This analysis showed a protein band of 3.3 kDa, identical to that of chemically synthesized TP-I, verifying that successful synthesis and secretion of TP-I by genetically engineered P. pastoris. The concentration of TP-I in the fermentation broth was 27.24-29.53 mg/L. High-resolution mass spectrometry analysis documented that the TP-I monomer had the same molecular weight, 2262.85, as the designed 17-amino acid sequence. The recombinant TP-I peptide displayed different levels of bactericidal activity against Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus. Thus, the present study demonstrated the feasibility of achieving high levels of expression of TP-I in P. pastoris.

[Liver dysfunction for 8 years with hypertension for 1 week in an 8-year-old girl].

A girl, aged 8 years, developed jaundice and liver dysfunction in the neonatal period, with congenital glaucoma diagnosed on day 5 after birth, hypertension and unusual facies (broad forehead, hypertelorism and deep-set eyes). Cholestasis was the main type of liver dysfunction. Cardiac macrovascular CTA showed stenosis at the abdominal aorta and the beginning of the bilateral renal arteries. Whole exon sequencing revealed a heterozygous frameshift mutation, c.1485delC (absence of cytosine), in exon 12 of the JAG1gene. The girl was diagnosed with Alagille syndrome and was given transaminase-lowering, cholagogic and antihypertensive treatment with multiple drugs. There were significant reductions in serum levels of alanine aminotransferase, aspartate aminotransferase and total bile acid, but blood pressure fluctuated between 102-140 mm Hg/53-89 mm Hg. After renal artery angiography and balloon dilatation angioplasty, the girl was given oral administration of antihypertensive drugs, and blood pressure was controlled at a level of 110-120 mm Hg/60-80 mm Hg. The rare disease Alagille syndrome should be considered when a child has refractory hypertension with the involvement of multiple systems, especially liver dysfunction with cholestasis as the main manifestation. Genetic causes should be analyzed for a early diagnosis.

Glucocorticoids Modulate Th1 and Th2 Responses in Asthmatic Mouse Models by Inhibition of Notch1 Signaling.

BACKGROUND: Notch1 has been linked to the pathogenesis of asthma due to its contribution on Th1/Th2 imbalance. γ-Secretase inhibitor (GSI) acts as an effective blocker of Notch1 signaling. Glucocorticoids (GCs) are the most effective anti-inflammatory drugs for asthma. The present study investigated the involvement of the Notch1 pathway in the anti-inflammatory effect of GCs and its association with Th1/Th2 balance. METHODS: The asthma model was established in BALB/c mice by sensitization with ovalbumin (OVA). Dexamethasone (DEX; 1 mg/kg) and/or GSI (0.03 mg/kg) was orally or intranasally administrated. RESULTS: Compared to the OVA-sensitized mice, the administration of DEX and/or GSI significantly ameliorated the airway inflammation infiltration, goblet cell metaplasia, and airway hyper-responsiveness. The expression of IL-4 and IL-13, as well as the ratios of eosinophils and lymphocytes, were significantly decreased, whereas IFN-γ and IL-2 levels were significantly increased in bronchoalveolar lavage fluid after the administration of DEX and GSI. The expressions of the Notch1/NICD1 pathway were decreased after DEX and/or GSI administration in lung tissues, especially in CD4+ T cells. Also, a reduction of GATA3 and elevation of T-bet levels were correlated with the upregulation of Th1/Th2 ratios in lung tissues. CONCLUSIONS: Through the inhibition of Notch1 signaling, both GSI and GCs could regulate Th1/Th2 balance involved in allergic airway inflammation in OVA-induced asthma.

The Role of Nanotechnology in Food Safety: Current Status and Future Perspective.

Nanotechnology is an emerging science in food production and processing sector, yet the role of nanotechnology in food safety has not been comprehensively reviewed. This study reviewed the types, sources and mode of actions of the nanoparticles used in the food systems. Additionally, the effect of nanoparticles on animal health and safety of the products of animal origin was evaluated. Moreover, retention of nutritionally important nanoparticle minerals in the animal systems and foods of animal origins was analyzed. Furthermore, food safety was critically evaluated in terms of antioxidative ability, antibacterial properties, and toxicological studies. Finally, the scope of nanoparticle-based functional foods and shelf-life enhancement using active packaging was discussed. The article concluded that although significant research has been done on the use of nanoparticles in food systems, yet commercialization of nanoparticle-based foods needs further investigation.

A Review on NanoPCR: History, Mechanism and Applications.

Polymerase Chain Reaction (PCR) is one of the most common technologies used in many laboratories to produce millions of copies of targeted nucleic acid under in vitro conditions. However, PCR faces multiple challenges including limited availability of DNA in the sample, high GC contents of the template, low efficiency, and specificity in amplification. Moreover, some DNA fragments are very difficult to amplify due to their secondary structure and high melting temperature requirement. To overcome these challenges, many approaches including the application of PCR additives in PCR mixture; change in instrument design; optimization of PCR system by using the accurate concentration of magnesium ions, primers, and cycle number; enzyme modification; and setting up the new touchdown and nested PCR strategies have been adopted. Although these approaches have enriched the output of PCR, they are not all-purpose and optimization can be case dependent. Nanometer-sized materials (nanomaterials) have offered a possible solution to these problems as these materials have exceptional physio-chemical properties as compared to macroscopic materials. Among these nanomaterials, silicon-based materials, carbon-based materials, semiconductor quantum dots (QDs), and some metals are well-known PCR enhancer. Hence, new PCR has been designed to utilize the unique properties of nanomaterial and is known as nanomaterial-assisted PCR or simply nanoPCR. Results of many studies have shown that the combination of these nanomaterials and biomolecules can mimic the DNA replication process successfully as present in the living organism. In this review, we have discussed the role of these different nanomaterials one by one and also discussed the mechanisms through which these nanomaterials enhance the efficiency of PCR.

Effect of simvastatin on the SIRT2/NF-κB pathway in rats with acute pulmonary embolism.

CONTEXT: Statins have been widely used in acute pulmonary embolism (APE), while simvastatin has been well-established for the prevention of pulmonary hypertension, which was supposed to be an attractive recommendation for APE treatment. OBJECTIVE: The current article studies the effect of simvastatin on the SIRT2/NF-κB pathway in rats with APE. MATERIALS AND METHODS: Sprague-Dawley rats were divided into four groups (n = 24 per group): control group, rats were treated with saline once daily for 14 days before administration of saline (sham group) or a suspension of autologous emboli (APE group), or rats were treated with simvastatin (10 mg/kg) for 14 days before administration of autologous emboli (APE + simvastatin) group. The RVSP, mPAP and the arterial blood gas was analyzed. Besides, plasma inflammatory cytokines and MMPs levels, as well as the expression of SIRT2/NF-κB pathway were determined. RESULTS: Compared with the control and sham groups, the levels of mPAP (31.06 ± 3.47 mmHg), RVSP (35.12 ± 6.02 mmHg), A-aDO2 (33.14 ± 6.16 mmHg) and MMP-9 (6.89 ± 0.84 ng/mL) activity were significantly elevated, but PaO2 (66.87 ± 7.85 mmHg) was highly decreased in rats from APE group at 24 h after APE. Meanwhile, the inflammatory changes were aggravated by the enhanced levels of TNF-α (138.85 ± 22.69 pg/mL), IL-1ß (128.47 ± 22.14 pg/mL), IL-6 (103.16 ± 13.58 pg/mL) and IL-8 (179.28 ± 25.79 pg/mL), as well as increased NF-κB (5.29 ± 0.47 fold), but reduced SIRT2 (59 ± 6% reduction), and eNOS (61 ± 5% reduction) mRNA in APE rats. APE rats treated with simvastatin led to a significant opposite trend of the above indexes. CONCLUSIONS: Simvastatin protects against APE-induced pulmonary artery pressure, hypoxemia and inflammatory changes probably due to the regulation of SIRT2/NF-κB signalling pathway, which suggest that simvastatin may have promising protective effects in patients with APE.

A boosting approach for prediction of protein-RNA binding residues.

BACKGROUND: RNA binding proteins play important roles in post-transcriptional RNA processing and transcriptional regulation. Distinguishing the RNA-binding residues in proteins is crucial for understanding how protein and RNA recognize each other and function together as a complex. RESULTS: We propose PredRBR, an effectively computational approach to predict RNA-binding residues. PredRBR is built with gradient tree boosting and an optimal feature set selected from a large number of sequence and structure characteristics and two categories of structural neighborhood properties. In cross-validation experiments on the RBP170 data set show that PredRBR achieves an overall accuracy of 0.84, a sensitivity of 0.85, MCC of 0.55 and AUC of 0.92, which are significantly better than that of other widely used machine learning algorithms such as Support Vector Machine, Random Forest, and Adaboost. We further calculate the feature importance of different feature categories and find that structural neighborhood characteristics are critical in the recognization of RNA binding residues. Also, PredRBR yields significantly better prediction accuracy on an independent test set (RBP101) in comparison with other state-of-the-art methods. CONCLUSIONS: The superior performance over existing RNA-binding residue prediction methods indicates the importance of the gradient tree boosting algorithm combined with the optimal selected features.

Quantitative Proteomic Profiling of Tachyplesin I Targets in U251 Gliomaspheres.

Tachyplesin I is a cationic peptide isolated from hemocytes of the horseshoe crab and its anti-tumor activity has been demonstrated in several tumor cells. However, there is limited information providing the global effects and mechanisms of tachyplesin I on glioblastoma multiforme (GBM). Here, by using two complementary proteomic strategies (2D-DIGE and dimethyl isotope labeling-based shotgun proteomics), we explored the effect of tachyplesin I on the proteome of gliomaspheres, a three-dimensional growth model formed by a GBM cell line U251. In total, the expression levels of 192 proteins were found to be significantly altered by tachyplesin I treatment. Gene ontology (GO) analysis revealed that many of them were cytoskeleton proteins and lysosomal acid hydrolases, and the mostly altered biological process was related to cellular metabolism, especially glycolysis. Moreover, we built protein-protein interaction network of these proteins and suggested the important role of DNA topoisomerase 2-alpha (TOP2A) in the signal-transduction cascade of tachyplesin I. In conclusion, we propose that tachyplesin I might down-regulate cathepsins in lysosomes and up-regulate TOP2A to inhibit migration and promote apoptosis in glioma, thus contribute to its anti-tumor function. Our results suggest tachyplesin I is a potential candidate for treatment of glioma.

The effect of H19-miR-29b interaction on bleomycin-induced mouse model of idiopathic pulmonary fibrosis.

Pulmonary fibrosis, characterized by the destruction of lung tissue architecture and the formation of fibrous foci, currently has no satisfactory treatment. Emodin is a component of Chinese herb that has been reported to be medicable on pancreatic fibrosis and liver fibrosis. However, its role in pulmonary fibrosis has not been reported yet. In the present study, we investigated the hypothesis that H19 play a promotive role in bleomycin-induced epithelial-mesenchymal transition of alveolar epithelial cell, and H19 exerts its effect through miR-29b regulation. H19 expression was positively correlated with COL1A1 and Acta2 expression; H19 knockdown inhibited COL1A1 and Acta2 expression. Moreover, H19 interacted with miR-29b through directly binding to the 3'UTR; miR-29b inhibited COL1A1 expression by directly binding to the 3'UTR. In conclusion, we revealed the promotive effect of H19 on BLM-induced IPF, and demonstrated the mechanism by which H19/miR-29b interaction exerts its effect on regulating pulmonary fibrosis. The present study provided a potential therapy to treat IPF.

A rapid, simple and sensitive method for the determination and bioequivalence study of transdermal fentanyl in human plasma using liquid chromatography-electrospray ionization tandem mass spectrometry.

OBJECTIVE: Since the 1960s, fentanyl has been used to replace morphine nd other opioids due to its higher potency in the treatment of acute pain; since the 1990s, it has also been administrated to control chronic pain by using transdermal fentanyl device system. It is crucial and of utmost importance and crucial to validate a sensitive method for the quantification of transdermal fentanyl in human plasma. MATERIALS AND METHODS: A rapid, simple and sensitive high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method has been established and validated for the determination of transdermal fentanyl in human plasma using fentanyl-D5 as an internal standard (IS). Following liquid-liquid extraction (LLE) with n-hexane, the extracts were separated on a Thermo Hypersil ODS(C18) column (2.1 × 150 mm i.d., 5 µm) interfaced with a triple-quadrupole tandem mass spectrometer using positive electrospray ionization. RESULTS AND CONCLUSIONS: Quantification of fentanyl was carried out by multiple reaction monitoring (MRM) of the transitions at m/z 337.1→188.0 for fentanyl and 341.9→187.9 for IS. The lower limit of quantification was 9.75 pg×mL-1, and the test showed a linear range of 9.75 - 10,000 pg×mL-1. The validated method was subsequently applied to a bioequivalence (BE) study in 24 healthy Chinese volunteers by using transdermal fentanyl patches.

Label-free detection of DNA by combining gated mesoporous silica and catalytic signal amplification of platinum nanoparticles.

This article presents a simple label-free detection of nucleic acids by using Pt@mesoporousSiO2 as a "smart" reporter, whose pores are first capped by single-stranded (ss) probe DNA. The detection signal is then amplified using the TMB oxidation reaction catalysed by Pt NPs while hybridizing with the complementary ss target DNA, which makes the pores of mesoporous SiO2 open through hybridization.

Application of functional microsphere in human hepatitis B virus surface antigen detection.

A novel and simple emulsifier-free emulsion polymerization technique was developed for preparation of mono-dispersed amino functionalized polymer microspheres with well defined diameters (about 400 nm). Various characterization methods demonstrated that the obtained amino microspheres had a uniform size and good dispersity which were confirmed by scanning electron microscope (SEM). Zeta potential and Fourier transform infrared spectrometer (FT-IR) demonstrated that amino groups have been successfully introduced to the microsphere surface. These functionalized microspheres have been shown to be efficient and controllable carriers capable of immobilizing and enriching monoclonal antibodies. Moreover, a newest chemiluminescent enzyme-linked immunoassay (ELISA) approach has been developed for human Hepatitis B virus surface antigen (HBsAg) detection. HBsAg was sandwiched between goat anti-HBsAg polyclonal antibody and mouse anti-HBsAg antibody. Alkaline phosphatase (ALP) conjugated horse anti-mouse immunnogloblin was used to bond with monoclonal antibody. Finally, chemiluminesent (CL) signals were recorded after adding 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD) which was used as a chemiluminescent substrate reagent of ALP. This novel chemiluminescent ELISA assay was proved to be of excellent specificity and high sensitivity when using ALP and AMPPD luminescence systems for specific HBsAg detection.

Magnetic beads-based chemiluminescent assay for ultrasensitive detection of pseudorabies virus.

A rapid, ultrasensitive and economical Pseudorabies virus (PRV) detection system based on magnetic beads (MBs) and chemiluminescence was developed in this paper. The carboxyl functionalized MBs (MBs-COOH) were covalently coupled with aminated DNA probes for capturing PRV biotinylated amplicon, the product of polymerase chain reaction (PCR). Agarose gel electrophoresis analysis approved the reliability of biotinylated amplicon. The MBs composites were incubated with alkaline phosphatase labeled streptavidin (ALP-SA) and chemiluminescene was determined by subsequently adding 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD). The optimal conditions of the PRV detection method were 10 microM for probe concentration, 50 degrees C for hybridization temperature and 30 min for hybridization time. The limit of detection (LOD) was as low as 100 amol/5 pM of amplicon which proved that this approach for PRV detection was ultrasensitive.