METHODS: A set of primers and probe targeting rrs genes of 22 Leptospira spp. were designed and evaluated on 31 Leptospira isolates, 41 other organisms and 65 clinical samples from suspected patients.
RESULTS: The developed assay was able to detect as low as 20 fg Leptospira DNA per reaction (equivalent to approximately 4 copies) and showed high specificity against the tested leptospiral strains. No cross amplification was observed with the other organisms. During the evaluation of the confirmed clinical specimens, the developed assay was able to correctly identify all positive samples (n = 10/10). One amplification was observed in a negative sample (n = 1/55). The sequencing of the PCR product of the discordant sample revealed that the sequences were similar to those of L. interrogans and L. kirschneri.
CONCLUSION: The findings suggest that the developed Taqman qPCR assay is sensitive, specific and has potential to be applied in a larger subsequent study.
RESULTS: A higher sensitivity was observed in chromosomal DNA (MPRE-PCR assay), where this assay allows the detection of gelatin DNA at amounts as as low as 1 pg, whereas mitochondrial DNA (CBH-PCR assay) can only detect at levels down to 10 pg of gelatin DNA. When an analysis with commercial gelatin and gelatin capsule samples was conducted, the same result was observed, with a significantly more sensitive detection being provided by the repetitive element of chromosomal DNA.
CONCLUSION: The present study has established highly sensitive DNA-based porcine detection systems derived from chromosomal DNA that are feasible for highly processed products such as gelatin and gelatin capsules containing a minute amount of DNA. This sensitive detection method can also be implemented to assist the halal authentication process of various food products available on the market. © 2018 Society of Chemical Industry.
RESULTS: A specific assay was developed, since no amplification was observed in viral strains from the same family of Paramyxoviridae, such as canine distemper virus (CDV), Newcastle disease virus (NDV), and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 × 104 copies/μL with Cq value of 34.32 ± 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34-0.53% and 1.38-2.03%, respectively. In addition, the clinical sample evaluation using this assay showed a higher detection rate, with 25 (35.2%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR.
CONCLUSIONS: The TaqMan-based real-time RT-PCR assay targeting the N gene described in this study is more sensitive, specific, rapid, and reproducible compared to the conventional RT-PCR assay targeting the N gene, which could be used to detect early infection in cats.
RESULTS: Tumors with a variety of clinical and pathological characteristics were selected. Gene expression stability and the optimal number of reference genes for gene expression normalization were calculated. RPS5 and HNRNPH were highly stable among OS cell lines, while RPS5 and RPS19 were the best combination for primary tumors. Pairwise variation analysis recommended four and two reference genes for optimal normalization of the expression data of canine OS tumors and cell lines, respectively.
CONCLUSIONS: Appropriate combinations of reference genes are recommended to normalize mRNA levels in canine OS tumors and cell lines to facilitate standardized and reliable quantification of target gene expression, which is essential for investigating key genes involved in canine OS metastasis and for comparative biomarker discovery.
MATERIALS AND METHODS: A total of fifty teeth specimens (n=50) were inoculated with E. faecalis for 21 days. Specimens were divided into five groups (Group 1: Myrrh, Group 2: Neem, Group 3: Liquorice, Group 4: 2% CHX and Group 5: Saline (negative control)). The intracanal medicaments were packed inside the tooth. After 5 days, the remaining microbial load was determined by using real time PCR.
RESULTS: Threshold cycle (Ct) values of Myrrh extract, Neem extract, Liquorice Extract, 2% CHX and saline were found to be 30.94, 23.85, 21.38, 30.93 and 17.8 respectively.
CONCLUSION: Myrrh extract showed inhibition of E.faecalis equal to that of 2% CHX followed by Neem, Liquorice and Saline.