Table 1.
List of PCR and Q-PCR primers used in this study.
Fig 1.
Specificity and sensitivity of the new PCR sexing assay based on SWIM and 12S gene amplification.
(A) Different crude embryo extracts containing 1 ng to 1000 ng of DNA were tested. Female chicken are detected by the amplification of two amplicons, the SWIM female specific amplicon of 212 bp and the 12S reference amplicon of 131 bp. (B) SWIM/12S PCR amplification of 4 sexed chicken breeds; Shaver black, Dekalb white, Isa brown, Bovan brown; on embryo extracts containing 100 ng DNA (M: Male, F: Female, C: Negative control, Mr: 50 bp ladder markers). (C) Three typical fluorescence profiles from female (♀), male (♂) and negative control containing no DNA template (NC) obtained by microfluidic capillary electrophoresis analysis. The female samples generated two peaks (SWIM: 212 bp and 12S: 131 bp), while the male samples gave only one peak at 131 bp (12S). No significant signal was observed for the negative control.
Table 2.
PCR sexing accuracy at different development stages for various tissues.
Fig 2.
Performance of the newly designed assays in comparison with previously described chicken PCR sexing methods.
(A, C, E, G, H, I) Agarose gel electrophoresis of PCR amplification from extracts of ISA brown chicken embryo using different primer sets: A. SWIM/12S, C. CHD P2/P8, E. CHD 2250F/2718R, G. EE0.6/CPE, H. EE0.6/SINT, I. XhoI/18S. 1 to 500 ng of either female or male DNA was used in each reaction (M: Male. F: Female. C: Negative control. Mr: 50 bp ladder markers). (B, D, F) Labchip analysis of the corresponding PCR (one female and one male) with different set of primers: B. SWIM/12S, D. CHD 2250F/2718R, F. CHD P2/P8 (500ng DNA input).
Table 3.
The main general attributes of PCR methods used in the sex identification of birds.
Fig 3.
Robustness assessment of the SWIM/12S PCR assay. Analysis by capillary electrophoresis.
(A) Superposition of the 176 electropherograms corresponding to the analysis of the 176 chicken embryo crude extracts. 2 markers were used to normalize the migration time of the PCR products (M-1: Marker-1 at the beginning of the measurement, M-2: Marker-2 at 1500bp). (B) All values of males’ and females’ peaks were highlighted with orange and green point, respectively. All tested female samples generated two peaks (SWIM: 212 bp and 12S: 131 bp), while male samples gave only one peak at 131 bp (12S). The upper panel contains three boxplots representing the distribution of the data.
Fig 4.
Sensitivity of our DMRT/Xho-I Q-PCR assay.
(A-E) CPA and MCA analysis of known sex ISA brown chicken (Green: Females, Orange: Males). Chicken embryo extracts containing 1 to 1000 ng DNA were used here (A: 1 ng, B: 10 ng, C: 100 ng, D: 500 ng and E: 1000 ng). For CPA analysis, fluorescence intensity was measured at each cycle and is represented in the upper left graph for each DNA concentration. Melting curves were obtained between 65°C and 95°C. (F) Mean melting curve profiles for females (Green) and males (Orange) chicken at 1 to 1000 ng DNA content. Mean Tm values +/- standard deviation are shown on the graph.
Fig 5.
Robustness analysis of the DMRT/Xho-I Q-PCR assay.
176 chicken samples sexed by conventional PCR method with primers 2250F/2718R [6] were used. Among them, 97 males (♂- Orange) and 84 females (♀- Green) were tested. 2 negative controls containing no DNA were included (A, B); Crossing point analysis (CPA) (C, D); Melting curve analysis (MCA). (A) Fluorescence signal of Q-PCR amplification for males (Orange), females (Green) and negative controls (Grey). (B) Cp values analysis of the 176 samples. Boxplots represent the statistical distribution of females and males’ CPA. (C) Mean Tm curves for females and males samples. Statistical distribution was represented by mean value, first and third quartiles +/- Standard deviation. (D) Ratio of Tm peak value 84°C/79°C for the 176 samples.
Table 4.
Comparative analyses of the main advantages and limitations of existing Q-PCR sexing methods.