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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

The ALV-J gene copies, GAPDH copies and relative virus copies among different organs in CHN06 group, NX0101 group and Tumor case. Statistical analysis was made using Two-way ANOVA in GraphPad Prism 5. *P < 0.01, **P < 0.001. Error bars indicate SEM.
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Fig5: The ALV-J gene copies, GAPDH copies and relative virus copies among different organs in CHN06 group, NX0101 group and Tumor case. Statistical analysis was made using Two-way ANOVA in GraphPad Prism 5. *P < 0.01, **P < 0.001. Error bars indicate SEM.

Mentions: Tissue samples of infected groups evaluated both by routine PCR and virus culture isolation with p27 antigen detection were all positive while control groups were all negative (data not shown). In addition, there were three cases with tumors, including two cases infected with NX0101 and one with CHN06. According to the standard curve formula: y = −3.505561x + 33.417706, the lg value of the virus copies in various organ tissues infected with ALV-J could be calculated by measuring the Ct value. At the same time, the lg value of the GAPDH copies in various organ tissues could be calculated by measuring the Ct value in accordance with the standard curve formula: y = −3.133720x + 35.762341.Data are obtained from the average of multifarious organ tissues of each group. To compare ALV-J gene copies among different organs in three groups, relative virus copies could be calculated by measuring quotient of the lg value of the virus copies divided by the lg value of the GAPDH copies. As Figure 5 showed, ALV-J genes could be detected in all organs of each group. In the CHN06 group, the heart, kidney and bursa had the highest viral gene load, liver and spleen had a medium viral load, and the lung and sternum had the lowest load. In NX0101 group, the copies of ALV-J were respectively highest in heart, higher in lung, kidney and bursa, and lowest in liver, spleen and sternum. In the tumor case, the heart, lung and kidney had the highest viral gene load; liver, spleen and bursa had a medium viral load, and the sternum had the lowest load. Except in the lung, the virus copies of CHN06 group were higher than that of NX0101 group in various organ tissues. From the above results, the highest copies of ALV-J were found in the heart and kidney.Figure 5


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)

The ALV-J gene copies, GAPDH copies and relative virus copies among different organs in CHN06 group, NX0101 group and Tumor case. Statistical analysis was made using Two-way ANOVA in GraphPad Prism 5. *P < 0.01, **P < 0.001. Error bars indicate SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: The ALV-J gene copies, GAPDH copies and relative virus copies among different organs in CHN06 group, NX0101 group and Tumor case. Statistical analysis was made using Two-way ANOVA in GraphPad Prism 5. *P < 0.01, **P < 0.001. Error bars indicate SEM.
Mentions: Tissue samples of infected groups evaluated both by routine PCR and virus culture isolation with p27 antigen detection were all positive while control groups were all negative (data not shown). In addition, there were three cases with tumors, including two cases infected with NX0101 and one with CHN06. According to the standard curve formula: y = −3.505561x + 33.417706, the lg value of the virus copies in various organ tissues infected with ALV-J could be calculated by measuring the Ct value. At the same time, the lg value of the GAPDH copies in various organ tissues could be calculated by measuring the Ct value in accordance with the standard curve formula: y = −3.133720x + 35.762341.Data are obtained from the average of multifarious organ tissues of each group. To compare ALV-J gene copies among different organs in three groups, relative virus copies could be calculated by measuring quotient of the lg value of the virus copies divided by the lg value of the GAPDH copies. As Figure 5 showed, ALV-J genes could be detected in all organs of each group. In the CHN06 group, the heart, kidney and bursa had the highest viral gene load, liver and spleen had a medium viral load, and the lung and sternum had the lowest load. In NX0101 group, the copies of ALV-J were respectively highest in heart, higher in lung, kidney and bursa, and lowest in liver, spleen and sternum. In the tumor case, the heart, lung and kidney had the highest viral gene load; liver, spleen and bursa had a medium viral load, and the sternum had the lowest load. Except in the lung, the virus copies of CHN06 group were higher than that of NX0101 group in various organ tissues. From the above results, the highest copies of ALV-J were found in the heart and kidney.Figure 5

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