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Development and application of SYBR Green I real-time PCR assay for the separate detection of subgroup J Avian leukosis virus and multiplex detection of avian leukosis virus subgroups A and B.

Dai M, Feng M, Liu D, Cao W, Liao M - Virol. J. (2015)

Bottom Line: In addition, the coefficients of variation for intra- and inter-assay were both less than 5%.Forty clinical plasma samples were evaluated by real-time PCR, routine PCR, and virus culture with positive rates of 80% (32/40), 72.5% (29/40) and 62.5% (25/40), respectively.When the assay for detection of ALV-J was used to quantify the viral load of various organ tissues in chicken inoculated by ALV-J strains CHN06 and NX0101, the results exhibited that ALV-J genes could be detected in all organ tissues examined and the highest copies of ALV-J were mainly in heart and kidney samples at 30 weeks post-infection.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China. daimanman1229@163.com.

ABSTRACT

Background: Subgroup A, B, and J ALVs are the most prevalent avian leukosis virus (ALV). Our study attempted to develop two SYBR Green I-based real-time PCR (RT-PCR) assays for specific detection of ALV subgroup J (ALV-J) and multiplex detection of ALV subgroups A and B (ALV-A/B), respectively.

Results: The two assays showed high specificity for ALV-J and ALV-A/B and the sensitivity of the two assays was at least 100 times higher than that of the routine PCR assay. The minimum virus detection limit of virus culture, routine PCR and real-time PCR for detection of ALV-A strain was 10(3) TCID50 units, 10(2) TCID50 units and fewer than 10 TCID50 units, respectively. In addition, the coefficients of variation for intra- and inter-assay were both less than 5%. Forty clinical plasma samples were evaluated by real-time PCR, routine PCR, and virus culture with positive rates of 80% (32/40), 72.5% (29/40) and 62.5% (25/40), respectively. When the assay for detection of ALV-J was used to quantify the viral load of various organ tissues in chicken inoculated by ALV-J strains CHN06 and NX0101, the results exhibited that ALV-J genes could be detected in all organ tissues examined and the highest copies of ALV-J were mainly in heart and kidney samples at 30 weeks post-infection. Except in lung, the virus copies of CHN06 group were higher than that of NX0101 group in various organ tissues.

Conclusions: The SYBR Green I-based real-time RT-PCR assay provides a powerful tool for the detection of ALV and study of virus replication and infection.

No MeSH data available.


Related in: MedlinePlus

Standard curve of real-time PCR for the detection of ALV-J, ALV-A/B, and GAPDH. (A) ALV-J plasmid ranged from 102 to108 copies/μL, (B) ALV-A/B plasmid ranged from 102 to108 copies/μL, (C) GAPDH plasmid ranged from 103 to108 copies/μL.
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Fig1: Standard curve of real-time PCR for the detection of ALV-J, ALV-A/B, and GAPDH. (A) ALV-J plasmid ranged from 102 to108 copies/μL, (B) ALV-A/B plasmid ranged from 102 to108 copies/μL, (C) GAPDH plasmid ranged from 103 to108 copies/μL.

Mentions: Serial dilutions from 108 - 102 copies/μL of plasmid pMDA/B, and pMDJ and 108 - 103 copies/μL of plasmid pMDG were used to produce standard curves for the qRT-PCR. Threshold cycle (CT) values were plotted against the known copy numbers of the standard controls. The results showed that there was good correlation between copy number and CT value of pMDA/B (R2 = 0.996196, efficiency = 1.058), pMDJ (R2 = 0.993029, efficiency =0.9287) and pMDG (R2 = 0.999114, efficiency = 1.085), respectively (Figure 1).Figure 1


Development and application of SYBR Green I real-time PCR assay for the separate detection of subgroup J Avian leukosis virus and multiplex detection of avian leukosis virus subgroups A and B.

Dai M, Feng M, Liu D, Cao W, Liao M - Virol. J. (2015)

Standard curve of real-time PCR for the detection of ALV-J, ALV-A/B, and GAPDH. (A) ALV-J plasmid ranged from 102 to108 copies/μL, (B) ALV-A/B plasmid ranged from 102 to108 copies/μL, (C) GAPDH plasmid ranged from 103 to108 copies/μL.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4403717&req=5

Fig1: Standard curve of real-time PCR for the detection of ALV-J, ALV-A/B, and GAPDH. (A) ALV-J plasmid ranged from 102 to108 copies/μL, (B) ALV-A/B plasmid ranged from 102 to108 copies/μL, (C) GAPDH plasmid ranged from 103 to108 copies/μL.
Mentions: Serial dilutions from 108 - 102 copies/μL of plasmid pMDA/B, and pMDJ and 108 - 103 copies/μL of plasmid pMDG were used to produce standard curves for the qRT-PCR. Threshold cycle (CT) values were plotted against the known copy numbers of the standard controls. The results showed that there was good correlation between copy number and CT value of pMDA/B (R2 = 0.996196, efficiency = 1.058), pMDJ (R2 = 0.993029, efficiency =0.9287) and pMDG (R2 = 0.999114, efficiency = 1.085), respectively (Figure 1).Figure 1

Bottom Line: In addition, the coefficients of variation for intra- and inter-assay were both less than 5%.Forty clinical plasma samples were evaluated by real-time PCR, routine PCR, and virus culture with positive rates of 80% (32/40), 72.5% (29/40) and 62.5% (25/40), respectively.When the assay for detection of ALV-J was used to quantify the viral load of various organ tissues in chicken inoculated by ALV-J strains CHN06 and NX0101, the results exhibited that ALV-J genes could be detected in all organ tissues examined and the highest copies of ALV-J were mainly in heart and kidney samples at 30 weeks post-infection.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China. daimanman1229@163.com.

ABSTRACT

Background: Subgroup A, B, and J ALVs are the most prevalent avian leukosis virus (ALV). Our study attempted to develop two SYBR Green I-based real-time PCR (RT-PCR) assays for specific detection of ALV subgroup J (ALV-J) and multiplex detection of ALV subgroups A and B (ALV-A/B), respectively.

Results: The two assays showed high specificity for ALV-J and ALV-A/B and the sensitivity of the two assays was at least 100 times higher than that of the routine PCR assay. The minimum virus detection limit of virus culture, routine PCR and real-time PCR for detection of ALV-A strain was 10(3) TCID50 units, 10(2) TCID50 units and fewer than 10 TCID50 units, respectively. In addition, the coefficients of variation for intra- and inter-assay were both less than 5%. Forty clinical plasma samples were evaluated by real-time PCR, routine PCR, and virus culture with positive rates of 80% (32/40), 72.5% (29/40) and 62.5% (25/40), respectively. When the assay for detection of ALV-J was used to quantify the viral load of various organ tissues in chicken inoculated by ALV-J strains CHN06 and NX0101, the results exhibited that ALV-J genes could be detected in all organ tissues examined and the highest copies of ALV-J were mainly in heart and kidney samples at 30 weeks post-infection. Except in lung, the virus copies of CHN06 group were higher than that of NX0101 group in various organ tissues.

Conclusions: The SYBR Green I-based real-time RT-PCR assay provides a powerful tool for the detection of ALV and study of virus replication and infection.

No MeSH data available.


Related in: MedlinePlus