Figure 1.
Standard curve of TaqMan MGB probe real-time quantitative polymerase chain reaction (PCR) for Chinese sacbrood virus (CSBV).
DNA standard curves of CSBV TaqMan PCR assay using a FAM-MGB-labeled TaqMan probe obtained with a 10-fold serial dilution (5×108 to 5×103 DNA copies) of a 3.1-kb plasmid that included a 320-bp fragment located in the CSBV VP1 gene. The standard curve was obtained by linear regression analysis of the CT measured for each amplification (y-axis) vs. the log copy number for each standard dilution (x-axis). The slope of the standard curve (–3.47) and the correlation coefficient are indicated r2 = 0.99 (Y = –3.47X+34.60). The red box represents Ct.
Figure 2.
Sensitivity of the TaqMan MGB probe real-time quantitative polymerase chain reaction (PCR) for Chinese sacbrood virus (CSBV).
To evaluate the sensitivity of the TaqMan MGB probe real-time RT-PCR assay. Amplification of seven 10-fold dilutions of DNA plasmid gave a titer that ranged from 5×106 to 5×100 DNA copies per reaction mixture. The reactions were performed in triplicate. The limit of detection of the TaqMan MGB probe real-time PCR was 50 CSBV genome equivalent copies.
Figure 3.
Comparison of TaqMan PCR assay and reverse transcriptase polymerase chain reaction (RT-PCR) methods by testing collected clinical samples.
The 16 collected clinical samples were tested using the TaqMan RT-PCR and RT-PCR, respectively. (A) RT-PCR test results. (B) TaqMan RT-PCR test results. The LNQY-1, LNJZ-3, LNYK-4, JLHN, LNQY-5 and LNSZ-2 samples tested positive using conventional RT-PCR and the TaqMan RT-PCR method, but the LNYK-6 samples tested positive using the TaqMan RT-PCR method, however the samples were negative by electron microscopy and conventional RT-PCR. lane 1. DNA marker DL2000, lane 2. LNQY-1, lane 3. LNQY-5, lane 4. LNQY-2, lane 5. LNYK-3, lane 6. LNYK-4, lane 7. LNYK-6, lane 8. LNLZ-1, lane 9. LNLZ-3, lane 10 JLJA, lane 11. JLHN, lane 12. LNJZ-1, lane 13. LNJZ-3, lane 14 LNJZ-5, lane 15 LNSZ-2 lane 16. JLBC.
Table 1.
Reproducibility of TaqMan minor groove binder (MGB) probe fluorescence real-time quantitative polymerase chain reaction (PCR).
Figure 4.
Comparison of the specificity of Chinese sacbrood virus (CSBV) TaqMan PCR assay for CSBV detection.
Chinese sacbrood virus strains gave a positive reaction using the TaqMan PCR assay method, no cross-reactivity with other clinically related viruses, including deformed wing virus (DWV), black queen cell virus (BQCV), acute been paralysis virus (ABPV), and chronic bee paralysis virus (CBPV), was detected.
Table 2.
Detection of 37 Chinese sacbrood virus (CSBV) clinical samples: of TaqMan minor groove binder (MGB) probe fluorescence real-time quantitative PCR, reverse transcriptase polymerase chain reaction (RT-PCR) and electron microscopy methods.