Assay methods for MDV-1
A technology of MDV-1 and samples, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, allergic diseases, etc., can solve the problems of difficult to design q-PCR primers, and achieve the specificity of the detector Effect
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Embodiment 1
[0096] Example 1: Design of qPCR primers and probes
[0097] The MDV pp38 gene sequence showed two single nucleotide differences between the attenuated vaccines CVI988 and Md5 (a virulent strain) (Fig. 1). polymorphism (G / A) (Big bold underline) represents MDV-1 vaccine virus and all MDV-1 virulent viruses that we have sequences (RB1B MDV, 584A MDV, 595MDV, 684A MDV, Md5 MDV, HPRS-B14 MDV, JM102 MDV, 660AMDV, 675A MDV , 549MDV, 571MDV, C12-130MDV), which is the polymorphism contained in the specific probe. polymorphism (G / A) (underlined in black italics) is not consistently present in all virulent virus strains. Table 1 lists the primer sequences designed to cover the polymorphic region and two probes (pp38Vacc_G_JUP, specific to the attenuated vaccine CVI988; and pp38Vir_A_JUP(1), specific to the virulent virus). A third probe, pp38-total_JUP, was also designed, targeting the consensus region of pp38 of all MDV-1 strains. All three probes can be used in combination wit...
Embodiment 2
[0102] Example 2: Infection of Chicken Embryo Fibroblasts (CEF)
[0103] Chicken embryo fibroblasts (CEF) were infected with CVI988 vaccine strain or RB1B virulent strain of MDV-1. Five days after infection, serial dilutions were prepared from these cells for singleplex qPCR.
[0104] at 75cm2 CEF (chicken embryo fibroblast) cell monolayers were established in culture flasks and infected with approximately 1000 plaque forming units (pfu) of cell-associated virus. After 5 days of culture at 38°C, cells were harvested and DNA was prepared using phenol-chloroform extraction. The reaction volume is 25ul and contains: Absolute Blue qPCR mastermix (ABgene), each primer 400nM (ie Ovo primer + virus-specific primer), each probe 200nM (ie Ovo probe + virus-specific probe), about 100ng DNA samples. The reaction was carried out in an ABI7500 real-time PCR instrument, and the cycle parameters were as follows: 50°C for 2min, 95°C for 15min, followed by 40 cycles at 95°C (15sec) and 60°C...
Embodiment 3
[0105] Example 3: Specificity of qPCR: Single Reaction
[0106] Reactions were set up essentially as described previously (Baigent et al., 2005a), each reaction containing 'ABsolute q-PCR master mix' (ABgene, Epsom, UK), forward and reverse primers, and one of the probes . Use ABI The q-PCR system (Applied Biosystems, Foster City, CA, USA) amplified and detected the reaction product under the following conditions: 50°C for 2min, 95°C for 15min, followed by 95°C (15sec) and 60°C (1min) 40 loops. Table 2 shows the primer and probe combinations for each single qPCR.
[0107] Table 2
[0108]
[0109] The specificity of the probe was first evaluated using 10-fold serial dilutions of DNA prepared from chicken embryo fibroblasts (CEF) 5 days after infection with CVI988 or RB1B (a virulent strain of MDV-1).
[0110] figure 2 Fluorescence changes produced by (a) CVI988-specific probes and (b) virulent-specific probes are illustrated using nucleic acid templates from CVI988 ...
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