A kit and its preparation method
A kit and culture technology, applied in the field of porcine epidemic diarrhea virus detection kit and its preparation, can solve the problems of inability to distinguish, unfavorable rapid detection and distinction of porcine epidemic diarrhea virus, etc.
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Embodiment 1
[0051] Embodiment 1. Preparation of detection kit
[0052] 1.1 Primer synthesis
[0053] By comparing and analyzing the S1 gene of the classic strain of porcine epidemic diarrhea virus and the variant strain of porcine epidemic diarrhea virus in recent years, a pair of porcine epidemic diarrhea virus-specific primers were designed to amplify the 5' end of the S1 gene with a size of about It is a 540bp fragment, and can be used to identify porcine epidemic diarrhea virus and other pathogens by PCR reaction. Primer pairs and sequences are as follows:
[0054]
[0055] Through the above design, a pair of specific primers for porcine epidemic diarrhea virus were obtained, which were respectively dissolved in sterile double distilled water and stored at -20°C.
[0056] 1.2 Preparation of PCR reaction solution (20 parts)
[0057] The PCR reaction solution mainly includes: 40 μl of upstream primer PSF (concentration 10 μmoL / L), 40 μl of downstream primer PSR (concentration of 1...
Embodiment 2
[0064] Embodiment 2. The use of detection kit
[0065] 2.1 Preparation of viral genome template in samples
[0066] Extract and store the viral genome template according to the instructions of the viral genome RNA extraction kit from Tiangen Biochemical Technology Co., Ltd.
[0067] 2.2PCR amplification
[0068] Prepare a one-step PCR reaction system according to the reagents prepared in part 1.2 of Example 1. The reaction system is a 50 μL system, where each reaction system includes 25 μl of 2×PCR Master Mix, 2 μl of upstream primers and 2 μl of downstream primers , 19 μl of double distilled water and 2 μl of viral genome template. After the preparation is completed, place the reaction system in a PCR amplification instrument, set the reaction program as 42°C for 45 minutes, 95°C for 3 minutes, 95°C for 30 s, 55°C for 30 s, 72°C for 40 s, for 35 to 40 cycles. Cycle and incubate at 72°C for 5 minutes to obtain amplified products. Take 5 μl of PCR amplified products and unde...
Embodiment 3
[0073] Example 3. Detection Kit Detection and Identification, Differentiation of Existing Porcine Epidemic Diarrhea Virus
[0074] 3.1 Detection and identification of the strains used
[0075] Utilize the detection kit prepared in Example 1 to detect and identify the S1 protein gene sequences of multiple porcine epidemic diarrhea virus classic strains and variant strains, the selected porcine epidemic diarrhea virus classic strains and variant strain representative strains S1 gene sequence information is as follows:
[0076] Strain name source Login ID plant type CV777 Belgium, 1993 AF353511 classic strain Br1 / 87 Great Britain, 1987 Z25483 classic strain DR-13 Korea, 2007 JQ023161 classic strain SM98 South Korea GU937797 classic strain
[0077] BP-5 Japan AB548618 classic strain SD-M China JX560761 classic strain NK Japan AB548623 mutant strain Chinju99 South Korea AY1...
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