Detecting DNA of causative agent of Mannheimia haemolytica by isolating DNA of causative agent of infectious disease from material, setting up one-step PCR, using fragment of genome of causative agent, measuring, and interpreting results
	CHERNYKH O YU; KRIVONOS R A; SHEVCHENKO A A; SHEVCHENKO L V; KOSHCHAEV A G; MANAKOVA A I; YAKOVLENKO P P
	2023-06-13
专利权人	UNIV KUBAN STATE AGRARIAN (UYKU-Non-standard)
申请日期	2023-06-13
专利号	RU2023115556-A
成果简介	NOVELTY - Method for detecting DNA of the causative agent of Mannheimia haemolyticainvolves isolating DNA of the causative agent of an infectious disease from the material, setting up a one-step PCR with fluorescent detection with 40 cycles of real-time amplification using oligonucleotide primers, probes, fluorescent dyes that are specific for a region of the genome of the causative agent of an animal infectious disease, an internal control sample in the form of a suspension of bacteriophage T4, and a positive control sample in the form of a mixture containing fragments of the genomes of the causative agent of an animal infectious disease and bacteriophage T4 with the nucleotide sequence in the same volume ratio, using a fragment of the genome of the causative agent of M.haemolyticafor the positive control sample, using a fluorescent dye, measuring the accumulation of fluorescent signals in the channels, and interpreting the results. USE - The method is useful for detecting DNA of the causative agent of M.haemolytica. DETAILED DESCRIPTION - Method for detecting DNA of the causative agent of Mannheimia haemolyticainvolves isolating DNA of the causative agent of an infectious disease from the material, setting up a one-step PCR with fluorescent detection with 40 cycles of real-time amplification using oligonucleotide primers, probes, fluorescent dyes that are specific for a region of the genome of the causative agent of an animal infectious disease, an internal control sample in the form of a suspension of bacteriophage T4 with a concentration of 5x 103phage particles per 1 μ l, and a positive control sample in the form of a mixture containing fragments of the genomes of the causative agent of an animal infectious disease and bacteriophage T4 with the nucleotide sequence in the same volume ratio: T4-F-tacatataaatcacgcaaagc (SEQ ID NO: Not defined) forward primer, T4-R-tagtatggctaatcttattgg (SEQ ID NO: Not defined) reverse primer, and T4-P-fluorescein amidites (FAM)-cattggcactgaccgagttc-black hole Quencher(RTM: A non-fluorescent chromophore) 1 (BHQ1) (SEQ ID NO: Not defined) probe, using a fragment of the genome of the causative agent of M.haemolyticawith the nucleotide sequence: Mh-F ctaaccaggacaacccacta (SEQ ID NO: Not defined) forward primer, Mh-R agtttaagtagyaagcgtgttcc (SEQ ID NO: Not defined) reverse primer, and Mh-P ROX-ctaagactaaaatcggatagcctgaaacgc-BHQ2 (SEQ ID NO: Not defined) probe for the positive control sample, using a fluorescent dye i.e. ROX/orange for the DNA of the causative agent of M.haemolytica, measuring the accumulation of fluorescent signals in the channels of the corresponding fluorescent dyes for a specific signal for testing the presence of an infectious disease agent in an animal and in the FAM/green channel for an internal control sample, and interpreting the results based on the presence or absence of intersection of the fluorescence curve with the threshold line, where if the fluorescence signal accumulation curves appear before the 35th cycle, then the reaction result is considered positive, and if the curves do not cross the threshold line or cross it after the 35th cycle, then the reaction result is negative.
IPC 分类号	C12Q-001/68
国家	俄罗斯
专业领域	生物科学
语种	英语
成果类型	专利
文献类型	科技成果
条目标识符	http://119.78.100.226:8889/handle/3KE4DYBR/21638
专题	中国科学院新疆生态与地理研究所
作者单位	UNIV KUBAN STATE AGRARIAN (UYKU-Non-standard)
推荐引用方式 GB/T 7714	CHERNYKH O YU,KRIVONOS R A,SHEVCHENKO A A,et al. Detecting DNA of causative agent of Mannheimia haemolytica by isolating DNA of causative agent of infectious disease from material, setting up one-step PCR, using fragment of genome of causative agent, measuring, and interpreting results. RU2023115556-A[P]. 2023.

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