A novel quantitative real-time PCR with the GAPDH reference gene for peste des petits ruminants.

Peste des petits ruminants (PPR) is a serious acute, highly contagious disease caused by the peste des petits ruminants virus (PPRV). This study aims to establish a qRT-PCR assay with an internal amplification control for the rapid and accurate detection of PPRV. The primers and probes for PPRV N were based on the national standard of the diagnostic techniques for PPR of China, and a pair of primers and TaqMan probes for the internal reference gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was designed. Optimisation of the reaction conditions, specificity, sensitivity and reproducibility tests, and clinical sample detection were conducted. The results showed that the optimal primers and probe concentrations of PPRV were 0.4 µmol/l and 0.4 µmol/l, respectively, and were 0.4 µmol/l and 0.2 µmol/l for the reference gene GAPDH, respectively. The established method has no cross-reaction with other viruses. The minimum detection limit was 6.8 copies/µl for PPRV and 190 copies/µl for GAPDH. The coefficients of variation (CV%) of PPRV and GAPDH were both lower than 2%. The results suggest that the PPRV qRT-PCR method containing internal reference genes has strong specificity, high sensitivity, and good reproducibility. The addition of internal reference genes for the sample quality control improves the accuracy of the detection.

Current Knowledge on Epizootic Haemorrhagic Disease in China.

Epizootic haemorrhagic disease (EHD) is an infectious, non-contagious viral disease of ruminants caused by epizootic haemorrhagic disease virus (EHDV) and is transmitted by insects of the genus Culicoides. In 2008, EHD was listed on the World Organization for Animal Health (WOAH) list of notifiable terrestrial and aquatic animal diseases. This article reviews the distribution of EHD in China and relevant studies and proposes several suggestions for the prevention and control of EHD. There have been reports of positivity for serum antibodies against EHDV-1, EHDV-2, EHDV-5, EHDV-6, EHDV-7, EHDV-8 and EHDV-10 in China. Strains of EHDV-1, -5, -6, -7, -8 and -10 have been isolated, among which the Seg-2, Seg-3 and Seg-6 sequences of serotypes -5, -6, -7 and -10 belong to the eastern topotype. The emergence of western topotype Seg-2 in EHDV-1 strains indicates that EHDV-1 strains in China are reassortant strains of the western and eastern topotypes. A novel serotype strain of EHDV named YNDH/V079/2018 was isolated in 2018. Chinese scholars have successfully expressed the EHDV VP7 protein and developed a variety of ELISA detection methods, including antigen capture ELISA and competitive ELISA. A variety of EHDV nucleic acid detection methods, including RT-PCR and qRT-PCR, have also been developed. LAMP and the liquid chip detection technique are also available. To prevent and control EHD, several suggestions for controlling EHD transmission have been proposed based on the actual situation in China, including controlling the number of Culicoides, reducing contact between Culicoides and hosts, continued monitoring of EHDV and Culicoides in different areas of China and further development and application of basic and pioneering research related to EHD prevention and control.

Association of menopause symptoms with depressive symptom severity in a diverse community-based sample.

The Dallas Heart Study dataset was used to examine relationships between menopausal symptoms and depressive symptom severity in 384 women (37-73 years old) self-reporting as menopausal. Self-reported menopausal symptoms were grouped based on the Menopause-specific Quality of Life Questionnaire (MENQOL). Depressive symptom severity was assessed using the Quick Inventory of Depressive Symptomatology - Self-Report (QIDS-SR). The relationship between menopause symptom groups, ethnicity and QIDS-SR was evaluated using multiple linear regression. Endorsement of sexual symptoms was positively associated with QIDS-SR score (ß = .12, p = .031), suggesting that sexual dysfunction during menopause may be a predictor of underlying depressive symptoms.

Development of a Blocking ELISA Using a Monoclonal Antibody to a Dominant Epitope in Non-Structural Protein 3A of Foot-and-Mouth Disease Virus, as a Matching Test for a Negative-Marker Vaccine.

Foot-and-mouth disease (FMD) is a devastating animal disease. Strategies for differentiation of infected from vaccinated animals (DIVA) remain very important for controlling disease. Development of an epitope-deleted marker vaccine and accompanying diagnostic method will improve the efficiency of DIVA. Here, a monoclonal antibody (Mab) was found to recognize a conserved "AEKNPLE" epitope spanning amino acids 109-115 of non-structural protein (NSP) 3A of foot-and-mouth disease virus (FMDV; O/Tibet/CHA/99 strain), which could be deleted by a reverse-genetic procedure. In addition, a blocking ELISA was developed based on this Mab against NSP 3A, which could serve as a matching test for a negative-marker vaccine. The criterion of this blocking ELISA was determined by detecting panels of sera from different origins. The serum samples with a percentage inhibition (PI) equal or greater than 50% were considered to be from infected animals, and those with <50% PI were considered to be from non-infected animals. This test showed similar performance when compared with other 2 blocking ELISAs based on an anti-NSP 3B Mab. This is the first report of the DIVA test for an NSP antibody based on an Mab against the conserved and predominant "AEKNPLE" epitope in NSP 3A of FMDV.

[Determination of streptomycin and dihydrostreptomycin residues in tomato paste by tandem dual solid phase extraction column-liquid chromatography-tandem mass spectrometry].

The method was specifically developed for the simultaneous determination of streptomycin and dihydrostreptomycin residues in tomato paste by tandem dual solid phase extraction (SPE) column cleanup-liquid chromatography-tandem mass spectrometry. The residues were extracted from the samples with phosphate buffer solution (pH 4). The cleanup was performed by the way of dispersive solid phase extraction and tandem dual solid phase extraction column. The polar chromatographic column was used to complete the separation of the analytes under gradient elution and the analytes were detected in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI +). The external standard calibration curves were used for the quantification. The linear ranges were from 0.01 to 0.2 mg/L with a good linear relationship (r > 0.999) for streptomycin and dihydrostreptomycin. The limit of quantification (LOQs) was 0.02 mg/kg for the both analytes. The recovery range was from 71% to 101% with the relative standard deviations (RSDs) between 2.3% and 15%. It was indicated that this method is accurate, easier, more sensitive, and has a better purification effect in the monitoring and analysis. The method is accurate and specific to monitor and analyze of streptomycin and dihydrostreptomycin residues in tomato paste and its products.