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Primer combination, kit and detection method for detecting Babesia canis
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The technology of Babesia canis and primer combination is applied in the directions of biochemical equipment and methods, recombinant DNA technology, and the determination/inspection of microorganisms. , strong specificity, easy to use effect
Inactive Publication Date: 2016-08-24
HENAN UNIV OF SCI & TECH
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Smear staining microscopic examination has low requirements for detection equipment and is easy to operate, but the detection rate is low
The detection of LAMP technology is simple and fast, and the results can be directly observed with the naked eye, but it is prone to false positives, and it is difficult to identify the type of parasite
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Embodiment 1
[0048] In this embodiment, the primer combination used to detect Babesia canis consists of an outer primer and an inner primer, and the outer primer is:
[0055] The kit used to detect Babesia canis in this example includes a primer combination consisting of an outer primer and an inner primer, and Taq enzyme 100U, 10×PCR Buffer 500μl, 25mmol.L -1 Mg 2＋ Salt 500μL, 10mmol.L -1 mixdNTP100μL, 10pmol·μL -1 Primer combination (including internal and external primers forward and reverse) 100 μL each, standard 25 μL and kit reaction condition instructions.
[0063] The method for detecting Babesia canis in the present embodiment comprises the following steps:
[0064] (1) Using the DNA sample to be detected as a template, a primer combination is used for nested PCR amplification. The primer combination consists of an outer primer and an inner primer. The outer primer is:
[0065] Forward primer: 5'-GGCTTTCGGTGATTCATA-3',
[0066] Reverse primer: 5'-CCTTCCGTCAATTCCTTT-3',
[0067] The inner primers are:
[0068] Forward primer: 5'-TGGACCATTCAAGTTTCTG-3',
[0069] Reverse primer: 5'-TCGTCTTCGATCCCCTA-3';
[0070] The reaction system for the first PCR amplification is: 50 μL system, Taq enzyme 1U, 10×PCR Buffer 5 μL, 25 mmol / LMg 2＋ 4μL, 10mmol / L mix dNTP1μL, 10pmol / μL outer primer forward and reverse primers 1μL each, DNA sample to be detected 1μL, add ddH 2 From 0 to 50 μL, the reaction program is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 25 cycl...
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Abstract
The invention discloses a primer combination, a kit and a detection method for detecting babeisa canis, and belongs to the technical field of biological detection. A nested PCR primer combination is designed for 18s RNAgene sequence of babeisa canis. The primer combination has high primer specificity, is not interfered by other non-target genes, and can determine a transcriptional level of babeisa canis rapidly, efficiently and accurately. The method for qualitatively detecting babeisa canis is simple and accurate, can accurately detect DNA samples with the minimum copy number of 10 within 4 hours, and has high detection sensitivity. The sensitivity of the nested PCR is 1,000 times higher than that of common PCR in gradient detections for a standard sample under the same experimental conditions; and the sensitivity of the nested PCR is 3-10% higher than that of the common PCR in actual clinical detections.
Description
technical field [0001] The invention specifically relates to a primer combination for detecting Babesia canis, a kit containing the primer combination, and a method for using the primer combination to detect Babesia canis, belonging to the technical field of biological detection. Background technique [0002] Babesiosis is a blood-borne protozoan disease transmitted by ticks that occurs widely in various livestock. It parasitizes in red blood cells singly or in pairs in domestic animals, and may cause severe anemia and hemoglobinuria. There are three kinds of pathogens that cause babesiosis in dogs, namely Babesia canis, B.vitlli and B.gibsoni. Babesia canis is found in Europe, the Americas and India, Babesia veschneri is found in South America, and Babesia gizzardi is found in Asia. The main species prevalent in my country is Babesia gibsonii, which is widely distributed in the country at present. Where there are ticks breeding, the disease can occur in spring, summer and...
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