Detection method for reverse transcription recombinase-aided amplification of avian influenza virus subtypes H5, H7, and H9.

BACKGROUND: Avian influenza virus (AIV) not only causes huge economic losses to the poultry industry, but also threatens human health. Reverse transcription recombinase-aided amplification (RT-RAA) is a novel isothermal nucleic acid amplification technology. This study aimed to improve the detection efficiency of H5, H7, and H9 subtypes of AIV and detect the disease in time. This study established RT-RAA-LFD and real-time fluorescence RT-RAA (RF-RT-RAA) detection methods, which combined RT-RAA with lateral flow dipstick (LFD) and exo probe respectively, while primers and probes were designed based on the reaction principle of RT-RAA. RESULTS: The results showed that RT-RAA-LFD could specifically amplify H5, H7, and H9 subtypes of AIV at 37 °C, 18 min, 39 °C, 20 min, and 38 °C, 18 min, respectively. The sensitivity of all three subtypes for RT-RAA-LFD was 102 copies/µL, which was 10 ∼100 times higher than that of reverse transcription polymerase chain reaction (RT-PCR) agarose electrophoresis method. RF-RT-RAA could specifically amplify H5, H7, and H9 subtypes of AIV at 40 °C, 20 min, 38 °C, 16 min, and 39 °C, 17 min, respectively. The sensitivity of all three subtypes for RF-RT-RAA was 101 copies/µL, which was consistent with the results of real-time fluorescence quantification RT-PCR, and 100 ∼1000 times higher than that of RT-PCR-agarose electrophoresis method. The total coincidence rate of the two methods and RT-PCR-agarose electrophoresis in the detection of clinical samples was higher than 95%. CONCLUSIONS: RT-RAA-LFD and RF-RT-RAA were successfully established in this experiment, with quick response, simple operation, strong specificity, high sensitivity, good repeatability, and stability. They are suitable for the early and rapid diagnosis of Avian influenza and they have positive significance for the prevention, control of the disease, and public health safety.

Based on CRISPR-Cas13a system, to establish a rapid visual detection method for avian influenza viruses.

To rapidly, specifically, and sensitively detect avian influenza virus (AIV), this research established a visual detection method of recombinase-aided amplification (RAA) based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated proteins 13a (Cas13a) system. In this study, specific primers and CRISPR RNA (crRNA) were designed according to the conservative sequence of AIV Nucleprotein (NP) gene. RAA technology was used to amplify the target sequence, and the amplification products were visually detected by lateral flow dipstick (LFD). The specificity, sensitivity, and reproducibility of RAA-CRISPR-Cas13a-LFD were evaluated. At the same time, this method and polymerase chain reaction (PCR)-agarose electrophoresis method were used to detect clinical samples, and the coincidence rate of the two detection methods was calculated. The results showed that the RAA-CRISPR-Cas13a-LFD method could achieve specific amplification of the target gene fragments, and the detection results could be visually observed through the LFD. Meanwhile, there was no cross-reaction with infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), and Newcastle disease virus (NDV). The sensitivity reached 100 copies/µL, which was 1,000-fold higher than that of PCR-agarose electrophoresis method. The coincidence rate of clinical tests was 98.75 %, and the total reaction time was ~1 h. The RAA-CRISPR-Cas13a-LFD method established in this study had the advantages of rapid, simple, strong specificity, and high sensitivity, which provided a new visual method for AIV detection.

Recombinase-aided amplification-lateral flow dipstick assay-a specific and sensitive method for visual detection of avian infectious laryngotracheitis virus.

The purpose of this study was to explore a specific, simple, and sensitive method for diagnosis of avian infectious laryngotracheitis virus. Recombinase-aided amplification (RAA) and lateral flow dipstick (LFD) were combined for labeling the optimized RAA probe with 6-carboxyfluorescein (FAM) and the 5'-end of the downstream primer with biotin, respectively. By optimizing the reaction time, temperature, and primer concentration of RAA, a RAA-LFD assay, which could be used for detection of infectious laryngotracheitis, was established. After the specificity and sensitivity test, the target gene fragments could be amplified by RAA-LFD assay in 20 min under isothermal conditions (37°C), and the amplification products could be visually observed and determined by LFD within 3 min. There was no cross-reaction with nucleic acids of other avian pathogens, the lowest detectable limit of RAA-LFD was 102 copies/µL, and the sensitivity of this method was 100 times higher than that of conventional PCR with the lowest detectable limit of 104 copies/µL. The results showed that RAA-LFD assay was highly sensitive, easy to use, and more suitable for clinical detection.

Research Note: Rapid detection of avian infectious laryngotracheitis virus with real-time fluorescence-based recombinase-aided amplification.

In this study, specific primers and fluorescent probes were designed to target the thymidine kinase (TK) gene sequence of avian infectious laryngotracheitis virus (ILTV). Through specificity and sensitivity tests, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method for detecting ILTV was established. The results showed that the method was specific and could be used to accurately detect ILTV, and there was no cross-reaction with Newcastle disease virus (NDV), avian influenza virus (AIV), or infectious bronchitis virus (IBV). Real-time fluorescence-based recombinase-aided amplification had high sensitivity, and the lowest detectable limit (LDL) for ILTV could reach 10 copies/µL, 1,000 times more sensitive than conventional PCR (104 copies/µL), to rival that of real-time fluorescence-based quantitative PCR (RFQ-PCR) (10 copies/µL). This method and RFQ-PCR were used to detect 96 samples of chicken throat swabs with ILT initially diagnosed in clinic from the north of China, and the coincidence rate of the 2 methods was 100%. The RF-RAA reaction required only 20-30 minutes to completing, and its sensitivity was much higher than that of conventional PCR. Real-time fluorescence-based recombinase-aided amplification is similar to RFQ-PCR and has the advantages of specificity, sensitivity, and high efficiency, so it is suitable for early clinical detection and epidemiological investigation of ILTV.

Reverse transcription recombinase-aided amplification assay combined with a lateral flow dipstick for detection of avian infectious bronchitis virus.

The study was conducted to develop a specific, simple, and sensitive method for diagnosis of avian infectious bronchitis virus (IBV). In this experiment, the selected downstream primer was labeled with biotin and the 5' end of RAA probe was labeled with FAM by reverse transcription recombinase-aided amplification (RT-RAA) combined with lateral flow dipstick (LFD). A RT-RAA-LFD assay that could be used for detection of IBV was established after optimization of RT-RAA reaction time, reaction temperature, and primer concentration. This method did not need reverse transcription of IBV template under isothermal condition (37°C), the amplification of target gene fragments could be completed within only 24 min, and the amplification products could be visually observed and determined by LFD within 3 min. The specificity test demonstrated that there was no cross reaction with the nucleic acids of other similar common pathogens. The lowest detectable limit for IBV was 102 copies/µL, and this method was 100 times more sensitive than conventional PCR (104 copies/µL), as verified by sensitivity test. The results showed that RT-RAA-LFD assay with strong specificity and high sensitivity was simple and easy to operate, and could be used for rapid detection of IBV in clinical diagnosis.

Establishment and application of real-time fluorescence-based quantitative PCR for detection of infectious laryngotracheitis virus using SYBR Green I.

In this study, a pair of primers were designed and synthesized for the gB gene of infectious laryngotracheitis virus (ILTV) (GenBank accession number: EU104985). The recombinant plasmid was constructed as the positive reference material, and a real-time fluorescence-based quantitative PCR (RFQ-PCR) method was established to detect ILTV using synergy brands (SYBR) Green I. This method could detect 3.34 × 103 copies/µL viral nucleic acid in the initial template, the sensitivity of this method was higher than that of the conventional PCR, and the coefficient of variation (CV) in the repeatability test by this method was 3.35%. At the same time, the method was used to detect 14 suspected pathological samples for clinical analysis, and the results showed that 10 positive samples were detected, and the standard S-shaped curve was amplified. It was concluded that the RFQ-PCR method established in this study was highly sensitive, specific and repeatable, it was suitable for early clinical detection and epidemiological investigation of ILTV, and was significant in effectively controlling the occurrence and transmission of ILTV.

Protective effects of baicalin against bromocriptine induced abortion in mice.

The Chinese herbal medicine Huang Qin (Radix Scutellariae) had been used for restless fetus for hundreds of years in China, however, little attention had been given to the components of the herb, specifically its ability to exert abortion-preventing effects at the maternal fatal interface. The present study was carried out to investigate the protective effects of baicalin and the possible mechanisms on pregnancies. Baicalin (at 10, 20, and 50 mg/kg BW respectively) was gavaged to bromocriptine-treated mice from gestation day (GD) 1 through GD 7. Abortion rates were calculated and the changes of interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and progesterone were assayed on different gestation days. Results showed that the embryonic death rates were significantly decreased in groups supplemented with 20 or 50 mg/kg BW of baicalin, accompanied with reduced IFN-gamma and enhanced progesterone contents. Moreover, the highest levels of IFN-gamma appeared on GD 5 both in the control and in baicalin treated groups. It is concluded that baicalin can exert an anti-abortive effect by cutting down the production of IFN-gamma and elevating the levels of progesterone in a dose dependent manner and IFN-gamma is involved in an inflammatory reaction which is beneficial for a successful implantation.

Effects of Radix scutellariae and Rhizoma atractylodis on LPS-induced abortion and the uterine IL-10 contents in mice.

In the present study, lipopolysaccharide (LPS) was injected i.v. via the tail vein (0.1 microg per mouse) to induce abortion (embryo resorption) in Kunming mice. The interleukin 10 (IL-10) contents in the uterus were assayed by ELISA. The results revealed that the IL-10 level was significantly decreased in the LPS-induced abortion group of mice compared to the controls. Use of Pentoxifylline (PXF), or a combination of Radix scutellariae and Rhizoma atractylodis reversed the LPS effects: bringing down the fetal resorption rate, and increasing the IL-10 level significantly. The study indicates that the anti-abortive effects of PXF and the combination of Radix scutellariae and Rhizoma atractylodis are closely related to up-regulation of the Th2 cytokine IL-10 at the maternal fetal interface.