Development of Duplex LAMP Technique for Detection of Porcine Epidemic Diarrhea Virus (PEDV) and Porcine Circovirus Type 2 (PCV 2).

Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detection of PEDV and PCV2 co-infection could be completed within approximately 1.5 h, and only a simple heating block was required for isothermal amplification. The preliminary validation using 50 swine clinical samples with positive and negative PEDV and/or PCV2 revealed that the sensitivity, specificity, and accuracy of duplex LAMP-LFD were all 100% in comparison to conventional PCR and RT-PCR. Hence, this study suggests that duplex LAMP-LFD is a promising tool for the early detection and initial screening of PEDV and PCV2, which could be beneficial for prevention, planning, and epidemiological surveys of these diseases.

Association of Single Nucleotide Polymorphisms of Endothelin, Orexin and Vascular Endothelial Growth Factor Receptor Genes with Obstructive Sleep Apnea among Thai Ethnic.

Background: Obstructive sleep apnea (OSA) is a complex disorder characterized by repetitive collapse of upper airway during sleep which strongly influenced by genetic factors, especially those affect regulation of the sleep-wake cycle and endothelial function. Objective: This study investigated the association between single nucleotide polymorphisms (SNPs) in endothelin (EDNRA), orexin (OX1R, OX2R) and vascular endothelial growth factor (VEGFR1) receptor genes with risk of OSA in Thai population. Material and Method: All subjects were diagnosed by overnight polysomnography (PSG) before divided into OSA (59) and NOSA (60) groups based on their apnea-hypopnea index (AHI). Serum lipid levels were examined by using enzymatic colorimetric and homogeneous methods. DNAs were extracted and genotyped the SNPs by polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis. Genotype distribution were analyzed using Chi-square test of SPSS program version 15.0. Results: The triglycerides level of OSA patients was significantly higher than NOSA (p-value = 0.002). The SNPs in EDNRA (rs5335), OX1R (rs2271933), OX2R (rs2292040, rs10456182) and VEGFR1 (rs11149523) genes showed no association with OSA. However, the SNP (rs17675063) in EDNRA gene showed significant differences in genotype distribution in the subjects with and without OSA (p-value = 0.002, odds ratio = 3.29 and 95% CI = 1.86-5.82). Conclusion: Obstructive sleep apnea, Single nucleotide polymorphisms, Endothelin receptor type A, Orexin receptor 1, Orexin receptor 2, Vascular endothelial growth factor receptor type 1.

Detection of Pathogenic Leptospires by Loop-Mediated Isothermal Amplification Targeting LipL32 Gene.

BACKGROUND: Leptospirosis is a worldwide re-emerging infectious disease caused by pathogenic leptospires including Leptospira interrogans. OBJECTIVE: In the present study, a loop-mediated isothermal amplification (LAMP) was developed to detect L. interrogans using lipL32 as a gene target. MATERIAL AND METHOD: Four specific primers were designed based on the conserved region of lipL32 gene of various serovars ofpathogenic leptospires. LAMP reaction was performed at 65 °C for 1 hour The LAMP products were detected by agarose gel electrophoresis andfluorescence dye. RESULTS: The lipL32 LAMP assay showed highly specificity to the reference stains of L. interrogans serovar Autumnalis, Bataviae, Javanica, Pyrogenes, Icterohaemorrhagiae, and Saigon. No product was produced from non-pathogenic leptospire (L. biflexa), human, or Escherichia coli. The lower limit of detection analyzed by agarose gel electrophoresis andfluorescence dye visualization was 0.02 pg/µl which equivalent to 4 genomic equivalents/reaction. Moreover the clinical strain of leptospires including pathogenic and intermediate group of L. interrogans were detected by lipL32 LAMP CONCLUSION: The developed lipL32 LAMP is high specificity and sensitivity that can be applied to detect pathogenic leptospires in clinical samples.

Apolipoprotein E receptor 2 Gene Polymorphisms Associated with Dyslipidemia among Thai Population.

BACKGROUND: Dyslipidemia is an abnormal amount of lipids and/or lipoproteins in the blood. It is a major risk factor of coronary heart disease and atherosclerosis. OBJECTIVE: This study investigated two single nucleotide polymorphisms (SNPs) in the apolipoprotein E receptor 2 (ApoER2) gene in association with risk of dyslipidemia in the Thai patients. MATERIAL AND METHOD: Four hundred blood samples including dyslipidemia patient (200) and unrelated normal control (200) were included in this study. Serum lipids were examined. DNAs were extracted and genotyped by using polymerase chain reaction (PCR) followed by high-resolution melting (HRM) analysis. The differences in genotype distribution between patient and normal control were assessed by Chi-square test of the SPSS software version 11.5. RESULTS: The data analysis revealed that two SNPs (rs3737984 and rs2297660) in ApoER2 gene had significant association with dyslipidemia. The rs3 737984 showed significant association at p-value = 0.001, in which A alleles informed the decreased risk of dyslipidemia [odds ratio and 95% CI of A allele, 0.42 (0.28-0.65)]. In contrast, the rs2297660 exhibited strongest association with an increase risk ofdyslipidemia [p-value = 0.001, odds ratio and 95% CI for theA allele was 2.38 (1.49-3.80)]. CONCLUSION: The rs2297660 may be used as biomarker for the risk of dyslipidemia in Thai ethnic.