Optimization and application of loop-mediated isothermal amplification technique for sex identification in red-whiskered bulbul (Pycnonotus jocosus)

Abstract

The red-whiskered bulbul (Pycnonotus jocosus) is a popular avian species in Thailand and many other countries. The red-whiskered bulbul has a high economic value, but breeding is challenging since sex identification is difficult. The PCR method is now used for sex identification. However, PCR amplification and post-PCR analysis necessitate the use of a laboratory equipped with specialized scientific instruments, which is inconvenient for field operations. This research describes a method for amplification of DNA samples using the loop-mediated isothermal amplification (LAMP) approach, which is a molecular biology methodology for isothermal amplification that is extremely sensitive, fast, and easy for post-LAMP product visualization. Herein, total of 23 blood samples were collected and DNA was extracted. Two sets of LAMP primers were designed for CHD-Z and CHD-W genes. The colorimetric assay was used to investigate the best conditions for LAMP reactions and post-LAMP product visualization. LAMP reactions for sex identification were compared to traditional PCR in terms of sensitivity and specificity. LAMP reactions were found to be 10-fold more sensitive than PCR at 1 ng of DNA. When compared to electrophoresis analysis, the visualization with colorimetric assay using GelRed® and SYTO™ 9 was 100% accurate. The optimal LAMP condition tested simple DNA extracted from bird feathers using the HotSHOT technique. The result showed that the optimal condition could distinguish the sex of red-whiskered bulbuls totally and accurately. A powerful method for red-whiskered bulbul sex identification is demonstrated in this study, which can be used in field studies because it is quick and easy to perform, has high sensitivity, and does not require advanced scientific equipment.

Keywords: LAMP; colorimetric assay; red‐whiskered bulbul; sex identification.

FIGURE 1

Red‐whiskered bulbuls (Pycnonotus jocosus) at Khao Yai National Park, Nakhon Ratchasima, Thailand, in a winter season.

FIGURE 2

Schematic diagram of LAMP primer sets designed for CHD‐Z and CHD‐W genes. Six different regions were designed for four primers: two outer primers (F3 and B3) and two inner primers (FIP and BIP) were generated from the inner regions joined by the TTTT‐linker. The FIP primer pairs are F1c and F2 and the BIP primer pairs are B2 and B1c.

FIGURE 3

The location of the candidates of outer primers (F3.1‐F3.3/B3) along the alignment of CHD‐Z and CHD‐W gene. The red letter represent a nucleotide base, which is different between the CHD‐Z gene and the CHD‐W gene.

FIGURE 4

The result of the PCR amplification of CHD‐W gene using the candidate outer primer F3.1/B3, F3.2/B3, and F3.3/B3. PCR products are amplified DNA are shown as differences between male and female for F3.2/B3.

FIGURE 5

The locations of LAMP primers on the CHD‐W and CHD‐Z gene of the red‐whiskered bulbul. The upper sequence is the CHD‐W gene, while the lower sequence is the CHD‐Z gene.

FIGURE 6

Comparative sex identification of red‐whickered bulbuls between PCR (a) and Post‐LAMP analysis method using electrophoresis (b), colorimetric assay using GelRed® (c), SYTO™ 9 (d), and CuSO₄ (e).

FIGURE 7

Agarose gel electrophoresis of female (a) and male (b) PCR products generated from primers P2/P8. DNA concentrations were amplified ranging from 10³ to 10⁻⁶ ng.

FIGURE 8

Sensitivity of post‐LAMP analysis method compared with electrophoresis using DNA concentration between 10³–10⁻⁶ ng