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Detection reagent of listeria monocytogenes and campylobacter fetus, and application thereof
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A technology for mononucleosis and Listeria, applied in the detection/testing of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc., can solve the problems of high cost and poor specificity, and achieve strong specificity, High sensitivity and high amplification efficiency
Inactive Publication Date: 2013-04-24
SHIHEZI UNIVERSITY
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Although the ELISA method has high sensitivity, its disadvantage is that the specificity is poor when the detection sample is contaminated; although the ELFA method has high specificity and sensitivity, the disadvantage of this method is that the cost is high
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Embodiment 1
[0046] refer to figure 1 — Figure 5 , this embodiment is divided into the following steps:
[0047] 1. Selection of conserved target sequences of the InlJ gene of Listeria monocytogenes and the 16S rDNA gene of Campylobacter fetalis
[0048] Download the published Listeria InlJ gene (GenBank accession numbers: EU817301-EU817320, EU262930-EU262933, etc.) AB301967, etc.), after converting it into a file with an extension of .seq, DNAstar was used to perform sequence comparison, and a highly conserved sequence without gene variation, insertion, and deletion was selected as the target sequence conserved for the two genes.
[0049] 2. Design and synthesis of specific primers
[0050] The target sequences of the selected Listeria monocytogenes InlJ gene and the 16S rDNA gene of Campylobacter fetalis were input into the primer premier 5.0 software program, and specific primers for the two genes were designed respectively. The specific requirements for primer design of InlJ gen...
Embodiment 2
[0067] This embodiment differs from Embodiment 1 in that:
[0068] 1. Standardization of duplex PCR reagents
[0069] According to the above test results, the reagents were standardized according to the optimization of the reaction conditions. The standardized reagent composition is:
[0072] 2.1 Sample processing: Take a certain amount of aborted fetus or placental disease material, first cut it into pieces with scissors, then grind it vigorously with a test tube grinder until it becomes a paste, add an appropriate amount of sterilized PBS, and pour ...
Embodiment 3
[0080] Compared with Example 2, this example differs in that the established duplex PCR detection method was used to detect Listeria monocytogenes and Campylobacter fetalis on 27 clinical samples of cattle and sheep abortion. Among them, 3 samples contained Listeria monocytogenes ( Figure 5 Lanes 3, 6, and 27), 1 sample contained Campylobacter fetalis (lane 16 in the figure).
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Abstract
The invention discloses a detection reagent of listeria monocytogenes and campylobacter fetus. The reagent comprises two pairs of specific primers which can amplify DNA genes of listeria monocytogenes InlJ and campylobacter fetus 16Sr. The invention discloses a preparation method of the reagent. The method comprises steps that: listeria monocytogenes InlJ and campylobacter fetus 16Sr DNA gene consensus sequences are selected, and specific primers are designed and synthesized. The invention also discloses an application of the reagent. The application method comprises steps of sample treatment, disease sample DNA extraction, double PCR reaction, and result determination. The molecular detection reagent of miscarriage caused by listeria monocytogenes and campylobacter fetus and the application thereof provided by the invention have advantages of high speed, good specificity, high sensitivity, and good repeatability. When the reagent is used in combination with a PCR technology, miscarriage caused by listeria monocytogenes and campylobacter fetus can be identified rapidly and effectively. Therefore, advantages of time saving and effort saving are provided.
Description
technical field [0001] The present invention relates to a molecular detection technology capable of distinguishing Listeria monocytogenes and Campylobacter fetal abortion, and in particular provides a molecular detection technology capable of distinguishing Listeria monocytogenes and Campylobacter fetal abortion The reagent and its preparation method and application belong to the technical field of animal food pathogen detection. Background technique [0002] Abortion in pregnant dams is the most common ailment in veterinary practice. In the practice of animal husbandry, there are many causes of abortion in cows and ewes, including genetics, climate and environment, endocrine, microbes, and feeding management, among which abortion caused by pathogenic microorganisms is an important factor. Listeria monocytogenes ( Listeria monocytogenes , LM) is a zoonotic pathogen that can cause listeriosis in humans and animals. Since the bacteria was first isolated in 1926, it has been...
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