To establish a new high resolution melting analytical method for identification of Lonicera japonica germplasm, the screening of 7 pairs of SSR (simple sequence repeats) primers, determining the suitable diagnostic primers by the differences of peak pattern and Tm was conducted. Then into the DNA template concentration, annealing temperature and the suitable range of cycle number were investigated. Combined with SIMCA-P software for data processing analysis, the results show that three main germplasm honeysuckle could be divided by four sets of primers. It provides methodology for improving L. japonica germplasm identification.

H5 subtype avian influenza (AIV-H5) is a major causative agent of animalloimia a rapid and sensitive molecular biological diagnosis is crucial to the control program of AIV-H5. AIV-H5 real-time fluorescent reverse transcription loop-mediated isothermal amplification (qRT-LAMP) was established by means of heat treatment of the samples. The sensitivity, specificity and repeatability of this method were assessed and the performance of Calcein,SYBR Green I,HNB,SYTO 81 in colorimetric detection was comparatively analyzed to screen the optimum dye. The results showed the sensitivity of this method was 100 times higher than that of standard real-time fluorescent RT-PCR, and the detection limit was one copy of the gene per reaction. This method had no cross-reactivity with other common avian respiratory tract infectious disease-related pathogens such as IBV and NDV. The present study suggested Calcein was the optimum dye. Small-scale tests suggested this method was reliable for survey monitoring of AIV-H5 on the spot, indicating its potential applications in field investigation.
Animals ; Chickens ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Influenza A Virus, H5N1 Subtype ; genetics ; isolation & purification ; Influenza in Birds ; diagnosis ; virology ; Poultry Diseases ; diagnosis ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; instrumentation ; methods ; Sensitivity and Specificity