Multiplex RT-PCR detection kit for porcine epidemic diarrhea viruses
A porcine epidemic diarrhea and RT-PCR technology, applied in the field of biological monitoring, can solve the problems of breeding loss, piglet diarrhea, etc., and achieve the effect of saving time and steps, high specificity, and reducing detection costs.
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Embodiment 1
[0029] The establishment of embodiment 1RT-PCR reaction system
[0030] 1) Three pairs of primers were designed according to the PEDV sequence (cv777) S1 gene, PSV sequence (YC2011) conserved gene and SAV sequence Cowden (AF182760) VP1 gene included in GenBank (Table 1).
[0031] Table 1 Multiplex RT-PCR primer design results
[0032]
[0033] 2) Treatment of sick materials
[0034] Use 10mmol·L -1 Dilute the feces sample 10 times with PBS buffer, shake and mix on the vortex for 5min, 12000r·min -1 Centrifuge for 5 minutes, and take the supernatant for later use.
[0035] 3) Extraction of RNA
[0036] Take 200μl sample and add 1ml RNAisoPlus, shake vigorously, let stand at room temperature for 5min; add 200μl chloroform, shake vigorously, let stand on ice for 10min, 4°C, 12000r min -1 Centrifuge for 15 minutes; after centrifugation, take 400 μl of the supernatant and add an equal volume of isopropanol, mix gently, and let stand on ice for 10 minutes, 4°C, 12000r·min -...
Embodiment 2
[0045] Embodiment 2 multiplex PCR specificity test
[0046] The mixed RNA templates of PEDV, PSV and SAV, RNA of PEDV-TGEV-GARV triple attenuated strains, RNA templates of PSV, SAV, CSFV, PRRSV, DNA templates of PCV2, PRV, PPV and H2O were used for multiple PCR reactions. The reaction system of template DNA is: TaKaRaExTaqHotStart (5units / μl) 0.25μl, 10×ExBuffer 5μl, dNTPMix (2.5mMeach) 4μl, upstream primer and downstream primer (10μM) each 1μl, template DNA 5μl and add DEPC water to 50μl. The reaction parameters are: 94°C for 3 min; 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec; and 72°C for 10 min after the cycle.
[0047] PEDV, PSV and SAV mixed RNA templates, PEDV-TGEV-GARV triple attenuated vaccine RNA, PSV, SAV, CSFV, PRRSV RNA templates, PCV2, PRV, PPV DNA templates and H 2 O for multiplex PCR amplification, the results are as follows Figure 4 As shown, only PEDV, PSV, and SAV mixed RNA templates and PEDV, PSV, and SAV RNA templates amplified ban...
Embodiment 3
[0048] The sensitivity of embodiment 3 multiplex RT-PCR
[0049] The three RNA concentrations of PEDV, PSV and SAV were 86 μg·mL -1 , 41μg·mL -1 and 0.72 μg·mL -1 , under the optimized RT-PCR reaction conditions, the above template RNAs were made 10-fold ratio (10 0 ~10 7 ) dilution, the result is as Figure 5 As shown, the highest sensitivity of PEDV is 43pg, the highest sensitivity of PSV is 21pg, and the highest sensitivity of SAV is 36pg.
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