Rapid visual detection of Mycobacterium avium subsp. paratuberculosis by recombinase polymerase amplification combined with a lateral flow dipstick

Paratuberculosis (Johne's disease) is a chronic debilitating disease of domestic and wild ruminants. However, widespread point-of-care testing is infrequent due to the lack of a robust method. The isothermal recombinase polymerase amplification (RPA) technique has applied for rapid diagnosis. Herein, RPA combined with a lateral flow dipstick (LFD) assay was developed to estimate DNA from Mycobacterium avium subsp. paratuberculosis. First, analytical specificity and sensitivity of the RPA-nfo primer and probe sets were assessed. The assay successfully detected M. paratuberculosis DNA in 30 min at 39℃ with a detection limit of up to eight copies per reaction, which was equivalent to that of the real-time quantitative polymerase chain reaction (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium spp. or five pathogenic enteric bacteria. Six hundred-twelve clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR, and enzyme-linked immunosorbent assay, respectively. The RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR results. This is the first report utilizing an RPA-LFD assay to visualize and rapidly detect M. paratuberculosis. Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource-constrained settings.

Association of rs3660 Single Nucleotide Polymorphisms with Non-Hodgkin's Lymphoma / 中国医科大学学报

Objective To evaluate the effect of single-nucleotide polymorphisms at the miRNA binding site rs3660 in the 3'-untranslated region of the KRT81 gene (miR-SNPs) on the cancer risk and clinical prognosis of non-Hodgkin's lymphomas (NHL).Methods The single-nucleo-tide polymorphisms of rs3660 was genotyped with ligation detection reaction method.The association of rs3660 with NHL survival was calculated with log-rank test using Kaplan-Meier method.Multivariate survival analysis was performed using a Cox proportional hazards model.Results The rs3660 genotype distribution difference was not statistically significant between the case and control group (P =0.50).Patients carrying the rs3660 CG/CC genotype exhibited a significantly longer survival time than patients carrying the GG genotype (P =0.012).In addition,rs3660 was associated independently with the survival of NHL patients in multivariate analysis (RR=0.589,95％ CI:0.415-0.832,P =0.004).The association of this miR-SNP with NHL survival was further confirmed in the peripheral T cell lymphoma (PTCL) subtype.Conclusion Our results indicate that KRT81 rs3660 GG type is an independent prognostic marker in NHL.

Establishment and Preliminary Application of the SYBR Green I Real-time PCR Assay for Detection of the Bovine Enterovirus / 病毒学报

The bovine enterovirus (BEV) is a pathogen found the digestive tracts of cattle. Recently, the BEV was discovered in cattle in a province in China. A rapid and effective detection method for the BEV is essential. An assay was carried out using two specific primers designed to amplify a highly conserved sequence of the 3D gene. A recombinant plasmid containing the target gene 3D was constructed as a standard control. The limit of detection of the reaction was 7.13 x 10(1) plasmid copies/μL of initial templates, which was tenfold more sensitive than the conventional reverse-transcription-polymerase chain reaction (RT-PCR). Moreover, the assay was highly specific because all negative controls and other viruses of clinical relevance did not develop positive results. Assay performance on field samples was evaluated on 44 (41 diarrhea and 3 aerosol) samples and compared with the conventional RT-PCR assay. Sixteen diarrhea samples were positive (16/41, 39. 02%) and 3 aerosol samples were positive (3/3, 100%). Preliminary results for clinical detection showed that the SYBR Green I real-time PCR assay was highly sensitive, specific and reproducible. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications for epidemics and in BEV research.

Establishment of goat limbal stem cell strain expressing Venus fluorescent protein and construction of limbal epithelial sheets / 生物工程学报

The integrity and transparency of cornea plays a key role in vision. Limbal Stem Cells (LSCs) are precursors of cornea, which are responsible for self-renewal and replenishing corneal epithelium. Though it is successful to cell replacement therapy for impairing ocular surface by Limbal Stem Cell Transplantation (LSCT), the mechanism of renew is unclear after LSCT. To real time follow-up the migration and differentiation of corneal transplanted epithelial cells after transplanting, we transfected venus (a fluorescent protein gene) into goat LSCs, selected with G418 and established a stable transfected cell line, named GLSC-V. These cells showed green fluorescence, and which could maintain for at least 3 months. GLSC-V also were positive for anti-P63 and anti-Integrinbeta1 antibody by immunofluorescent staining. We founded neither GLSC-V nor GLSCs expressed keratin3 (k3) and keratinl2 (k12). However, GLSC-V had higher levels in expression of p63, pcna and venus compared with GLSCs. Further, we cultivated the cells on denude amniotic membrane to construct tissue engineered fluorescent corneal epithelial sheets. Histology and HE staining showed that the constructed fluorescent corneal epithelial sheets consisted of 5-6 layers of epithelium. Only the lowest basal cells of fluorescent corneal epithelial sheets expressed P63 analyzed by immunofluorescence, but not superficial epithelial cells. These results showed that our constructed fluorescent corneal epithelial sheets were similar to the normal corneal epithelium in structure and morphology. This demonstrated that they could be transplanted for patents with corneal impair, also may provide a foundation for the study on the mechanisms of corneal epithelial cell regeneration after LSCT.