Multiple sequences alignment
From NCBI, 6 COI partial gene sequences of swiftlets belonging to the
Aerodramus genus were downloaded from the NCBI database with accession numbers KY350188.1, KY350193.1, KY350191.1, KY350190.1, JQ174502.1 and JQ173912.1. In addition, 2 COI partial sequences of
Collocalia spp. (JQ174497.1, JQ174500.1) and 2 sequences of
Apus spp. (KU722438.1, KY242302.1) were also selected for multiple sequence alignment to find the highly conserved region in
Aerodramus spp. and distinct from 2 other genera (
Collocalia spp. and
Apus spp.) in order to enable the design of a primer pair that amplify a short COI sequence from
Aerodramus spp. The results showed that there were several variative regions between
Aerodramus spp. and 2 other genera, specifically the position is from 52 bp to 91 bp and the position is from 201 bp to 230 bp potentially to design primers.
For ND2 gene region, 7 ND2 partial sequences of
Aerodramus spp. (EU085333.1, EU085332.1, KJ671363.1, KJ671358.1, KJ671355.1, KJ671344.1, KJ671340.1), 2 sequences of
Collocalia spp. (KF818946.1, KF818945.1) and 2 sequences of
Apus spp. (KR905656.1, KR905653.1) were obtained for multiple sequence alignment. The region from 231 bp to 383 bp was selected for primer design using Primer3 tool with forward primer was located from 231 bp to 290 bp and reverse primer was from 345 bp to 383 bp.
Cyt-b gene
9 sequences belonging to the genus
Aerodramus spp (MG653625.1, KJ671364.1, KJ671365.1, KJ671366.1, KJ671367.1, KJ671369.1, KJ671370.1, KJ671371.1, KJ671372.1, KJ671373.1, KJ671374.1, KJ671375.1, KJ671379.1, KJ671380.1, KJ671384.1, JN709906.1, JN709907.1, JN709922.1, JN709923.1), 3 sequences from the genus
Collocalia spp (JQ353853.1, MG322679.1, MG653638.1) and 2 sequences from the genus
Apus spp (JQ353923.1, JQ353899.1) were compared for sequence alignment. The gene segment ranging from position 489 to 625 bp was selected for primer design.
Fib7 gene
7 sequences from the genus
Aerodramus spp (AY513077.1, AY513075.1, AY513078.1, JQ520052.1, AY513098.1, AY513101.1, JQ520047.1), 2 sequences from the genus
Collocalia spp (JQ520050.1, JQ520049.1) and 2 sequences from the genus
Apus spp (JQ520068.1, JQ520069.1) were compared to identify suitable sequence regions for designing forward and reverse primers. The gene segment from position 565 to 749 bp was chosen for designing PCR amplification primers targeting the Fib7 gene sequence of the
Aerodramus spp swiftlets. The forward primer was designed based on the sequence region from position 565 to 583 bp, while the reverse primer was designed based on the sequence region from position 724 to 749 bp.
Primer design
The result of designing the COI primer pair provided by the Primer3 software (Table 3) most values meet the requirements for primer design, but in terms of primer efficiency, the self-dimer formation energy of the reverse primer (-9.89 kcal.mol
-1) does not satisfy the criterion of being greater than -9 kcal.mol-1. However, this value deviates only slightly from the requirement, so the primer pair is still used for specificity testing using the Primer Blast tool. Similarly, for the primer design results of the ND2, Cyt-b and Fib7 genes, primer pairs were selected to assess their specificity.
The results of the specificity test using the Blast software for all four primer pairs showed the ability to specifically bind with swiftlets belonging to
Aerodramus spp.
The optimization of the annealing temperature
The annealing temperature of the primer pair amplifying the COI gene: The results of the COI primer pair were shown in Fig 1. PCR product bands were successfully amplified at all tested temperatures, resulting in a 157 bp size. The primer pair consistently produced bright bands, but there were differences in band intensities across the tested temperature levels. The lowest intensity was observed at 55
oC, gradually increasing with higher annealing temperatures. However, at 60oC, the band intensity was higher than at 61
oC. Additionally, at an annealing temperature of 60
oC, the brightest band was obtained without any non-specific products or primer dimers. This temperature was chosen for the next experiment.
The annealing temperature of the primer pair amplifying the ND2 gene: the results of ND2 primers are shown in Fig 2 where PCR product bands at all tested annealing temperatures were amplified correctly at the expected size of 150 bp. All annealing temperatures produced bright bands without any non-specific products or primer-dimers. Among the tested temperatures, 60
oC and 61
oC exhibited better results compared to the other temperatures. but 60
oC was chosen for further investigation due to its smaller deviation from the average melting temperature provided by the software.
The annealing temperature of the primer pair amplifying the Cyt-b gene: The results of gradient annealing temperature survey for Cyt-b primer pairs were shown in Fig 3, the PCR product bands at all tested annealing temperatures were amplified at the correct size of 130 bp. There were no non-specific products or primer-dimers. Specifically, at 60
oC the band intensity is brightest. Therefore, the optimal annealing temperature for amplifying the Cytb gene using this primer pair is determined to be 60
oC and it was chosen for further investigation.
The annealing temperature of the primer pair amplifying the Fib7 gene: the results were shown in Fig 4, where all amplified product bands have a size of 173 bp. No non-specific products are observed, but primer-dimers are present. At 60
oC, the band intensity is relatively higher compared to 61
oC and it represents the highest intensity among the tested annealing temperatures. Therefore, the optimal annealing temperature for the AeroFib7f/AeroFib7r primer pair is determined to be 60
oC and it was selected for further investigation.
The sensitivity of primer pairs
The sensitivity analysis results of the COI primer pair: the results of the sensitivity assessment of the COI primer pair in Fig 5 showed that as the DNA concentration decreased from 100 times to 10000 times, the intensity of the band also decreased but was still visible. At a DNA concentration lower than 15x10
-5 ng/µL, the band of the amplified product became fainter and almost invisible. Therefore, the sensitivity of the reaction was determined to be 15´10-4 ng/µL.
The sensitivity analysis results of the ND2 primer pair: from Fig 6 the result of the sensitivity of ND2 primer pair, it can be observed that at the first dilution factor of DNA sample, no product band appeared, only the product was observed at the original DNA concentration with a size of 150 bp. The extended smeared bands indicate that the DNA concentration is too low compared to the sensitivity of the primers, causing the primer to fail to bind to the target sequence and amplify the product. Therefore, the sensitivity of the reaction is 15 ng/µL.
The sensitivity analysis results of the Cyt-b primer pairs: from Fig 7 (A), it can be seen that at the initial dilution factor 100 times, the Cytb primer pair was still able to amplify a clear and bright product band at 130 bp and the intensity of the band is not significantly different from that of the original DNA concentration. However, at the dilution factor of 1000 times, the product band becomes fainter, but still observable and at subsequent dilution factors, no product band is detected. Therefore, the sensitivity of the reaction is 15x10
-3 ng/µL.
The sensitivity analysis results of the Fib7 primer pair: From Fig 8 (B), it can be observed that at the original DNA concentration, a clear and bright product band is visible at 173 bp. At the first dilution factor, no product band is detected, but at the lower dilution of 1000 times and 10000 times, faint and indistinct bands can be observed. This could be due to the low initial DNA concentration, resulting in extremely low DNA amounts at dilutions of 100, 1000 and 10,000 times, with minimal differences, so the amount of DNA obtained during pipetting at different concentrations is random and lower concentrations tend to yield more DNA, resulting in the appearance of bands. This possibility is supported by the absence of bands in the negative control sample containing BiH2O, indicating no contamination between the dilutions. Hence, the sensitivity of the reaction is 15 ng/µL.
The results from multiple sequence aligment of the COI gene sequence align with
Hebert et al., (2003), suggesting that the COI mitochondrial gene exhibits high mutation rates and significant variation among avian species. Hence, the COI gene region is often suitable for identifying closely related species, such as swiftlet species belonging to the genera
Aerodramus spp.,
Collocalia spp. and
Apus spp., due to several appropriately varied sequence regions suitable for primer design. Moreover, selecting a shorter DNA sequence, approximately 100-300 bp, for primer design targeting easily degraded DNA would be more suitable and effective compared to longer sequences
(Dai et al., 2015). Similarly, the comparison of the ND2 gene sequences reveals the presence of conserved segments characteristic of higher-order species, along with specific variant positions that could be useful for species differentiation. This outcome aligns with previous analyses showing that the ND2 mitochondrial gene exhibits the highest nucleotide variability, accounting for 9.7% when compared to other genes in swiftlet samples
(Quek et al., 2018). Therefore, the mitochondrial ND2 gene sequence appears as a useful molecular marker capable of distinguishing closely related swiftlet species. The chosen sequence size for amplification falls within the range of 100–300 bp (from position 230 to 385 bp), is suitable for amplifying easily degraded DNA, such as from bird’s nests and is relatively shorter than the sequence chosen in a previous study differentiating the origins of bird nests in Thailand, where the selected region length was 356 bp
(Lv et al., 2021).
The primer pairs designed for the amplification of COI, ND2 and Cyt-B genes exhibited complete specificity towards the
Aerodramus genus. The primer pair targeting the Fib7 gene exhibits potential matches with some swiftlet species closely related within the Apodidae family. These species include
Collocalia linchi,
Collocalia esculenta,
Collocalia affinis,
Raphidura leucopygialis and
Hydrochous gigas, as indicated by NCBI Blast results, but this Fib7 primers did not match the DNA sequence of other organisms. Therefore, this lack of specificity does not compromise the study’s primary objective, which is to detect DNA from swiftlets capable of producing edible bird nests, notably associated with two prominent species,
Aerodramus fuciphagus and
Aerodramus maximus. Therefore, despite this limitation, this primer pair continues to be utilized in the experiments.
In the sensitivity analysis experiment, the sensitivity of mtDNA was definitely higher than the sensitivity of nDNA. This was also suitable with characteristics between two gene regions when the mtDNA existence in multiple copies in cells. Furthermore, nDNA analysis requires higher amount of template than mtDNA leading to less sensitivity of analysis
(Orbayinah et al., 2019).