| ObjectiveThe edible bird’s nest (EBN) is mainly composed of the saliva by several species of Aerodramus or Collocallia genus in the Apodidae family. EBN is predominately produced in Southeast Asia. Due to the rarity and supposedly exquisite flavor, EBN is expensive and fake or adulterated EBN is frequently found in the market. Therefore, there is urgent need to establish a simple and reliable method for the identification and evaluation of EBN. Recently, DNA barcode technology based on DNA sequencing has been widely used in the identification of Chinese herbal medicine for its accuracy and rapidity. Especially, real-time fluorescent quantitative PCR technology has distinct advantages due to the high efficiency and high sensitivity. The different kinds of EBN are produced by different species of swiftlets, and their price and quality are various. In order to understand the origin species of EBN produced in different areas, we collected 32 kinds of EBN with different varieties produced from different countries, and identified the species of EBN using DNA barcode technique. We aimed to investigate the phylogenetic relationships among the different varieties of EBN and facilitate the evaluation of EBN quality.Methods1. Total 32 kinds of EBN with different varieties produced from different countries, were collected, including 31 white or silver nests and 1 black nest. Total genomic DNA was isolated from the samples using commencial kits. Cytb gene sequences were amplified by using the primer ND5 (5’-TAGCTAGGATCTTTCGCCCT-3’) and H15709 (5’ GGCATATGCGAATARGAARTATCA-3’). PCR products were purified and subjected to bidirectional sequencing by ABI3730 sequencer. The results were blasted against GenBank databases using BLAST server provided by USA National Center for Biotechnology Information (NCBI) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The phylogenetic tree was constructed using Neighbor-joining (NJ) method.2. The primers for Cytb, ND2, COI and 12S rRNA genes were designed according to the sequences of Aerodramus downloaded from Genbank. PCR was perforemd to amplify these genes and the products were sequenced by ABI3730 sequencer. Identification of the original species was performed using similar BLAST from GenBank and method of nearest distance. The seqeunces were analzyed using Clustal WMEGA6.0 software. Genetic distance was calculated and Neighbor-joining (NJ) phylogenetic tree was constructed. DNA barcode of the original species of EBN was established based on Cytb, ND2, COI and12S rRNA genes.3. The sequences of cytochrome b gene of Aerodramus were compared to those of other animals to find a region that can distinguish the species and TaqMan probe and primers for real-time quantitative PCR were designed. The mitochondrial DNA was isolated from EBN samples using the method of Protease K and used as the template for real-time fluorescent quantitative PCR. The genome DNA extracted from EBN after purification were diluted in a series and used as positive standard, the standard curve was established using the logarithm of the standard template copy number as x-axis and the CT value of real-time fluorescence quantitative PCR as y-axis. The mitochondrial DNA of human, rat, pig, chicken, fish, seaweed were used as negative control to determine the specificity. Different concentrations of DNA template from EBN were used to investigate the sensitivity. Three concentrations of positive DNA template were detected 5 times to determine the repeatability and stability of this method.4. To identify whether the samples contained pigskin composition in EBN, pigskin was mixed with EBN samples with the proportion of 50%,30%,10%,5%, and 1%, respectively, total DNA was extracted and subjected to PCR analysis using porcine universal primers, PCR products were detected by agrose electrophoresis and sequenced to identify the species. Results 1. We selected Cytb sequence with the length of 290 bp after splicing, correction and alignment using MEGA software. BLAST results showed that among the dozens of similar sequences all of them were from the species of Aerodramus genus in the Apodidae family, and the most similar sequence similarity was 100%. From the Cytb gene sequences of 32 samples of EBN, we detected 290 conserved sites and 20 variable sites, conservative sites accounted for 93.1% of the total number of nucleotides and variable sites accounted for about 6.9% of the total number of nucleotides. K-2p genetic distance analysis showed that genetic distance among all the samples was in the range of 0.000-0.062. Based on genetic distance and parsimony informative sites of the 32 EBN samples, these samples were from three different original species. NJ phylogenetic analysis showed that 23 white or silver nests samples were clustered in Aerodramus fuciphagus, and the positive rate was 84%, so we concluded that their genetic origin was Aerodramus fuciphagus.8 white or silver nests samples were clustered in Aerodramus fuciphagus germani, and the positive rate was 74%, so we concluded that their genetic origin was Aerodramus fuciphagus germani. The black nest sample was clustered in Aerodramus maximus, and the positive rate was 96%. Because the sequences of Aerodramus maximus and its subspecies Aerodramus maximuslowi deposited in Genbank did not show differences, we concluded that its genetic origin was Aerodramus maximus or the subspecies Aerodramus maximuslowi.2. Next we analyzed 124 bp PCR products of ND2 from all EBN samples. BLAST results showed that the most similar sequences with all the samples of white or silver nests were from Aerodramus fuciphagus, and the highest similarity of 23 samples was 100%, while the highest similarity of 8 samples was 98%. The most similar sequence with the black nest sample was from Aerodramus maximus. NJ phylogenetic tree showed that 31 white or silver nests samples were clustered in Aerodramus fuciphagus and the positive rate was 62%, among which 8 samples (No.11,17,21,22,23,25,26 and 28) had a separate branch with a positive rate of 82%. No.32 sample was clustered in Aerodramus maximus and the positive rate was 72%. Comparison of ND2 sequences with those deposited in Genbank showed that there were 2 parsimony informative sites between Aerodramus fuciphagus and Aerodramus fuciphagus germani and 10 parsimony informative sites between Aerodramus fuciphagus and Aerodramus maximus, and different species of Aerodramus had several parsimony informative sites.3. We analyzed 182 bp PCR products of COI from 29 EBN samples. The results of BLAST and NJ phylogenetic analysis showed that the genetic origin of EBN was consistent with the results based on the analysis of Cytb and ND2 genes. All EBN samples from Aerodramus fuciphagus germani had a separate branch with a positive rate of 79%. The alignment of COI sequences showed that there were 2 parsimony informative sites between Aerodramus fuciphagus and Aerodramus fuciphagus germani and 9 parsimony informative sites between Aerodramus fuciphagus and Aerodramus maximus.4. We analyzed 560 bp PCR products of 12S rRNA from 22 EBN samples. The results of BLAST and NJ phylogenetic analysis showed that the genetic origin of EBN was consistent with the results based on the analysis of Cytb, ND2 and COI genes. All EBN samples from Aerodramus fuciphagus germani had a separate branch with a positive rate of 84%. The alignment of 12S rRNA sequences showed that there were 5 parsimony informative sites between Aerodramus fuciphagus and Aerodramus fuciphagus germani, and 9 parsimony informative sites between Aerodramus fuciphagus and Aerodramus maximus. In addition, there was 1 parsimony informative site within the Aerodramus fuciphagus samples and there was a base deletion in 12S rRNA sequence of Aerodramus maximus compared to Aerodramus fuciphagus. These results suggested that DNA barcode based on 12S rRNA may provide more information on the identification of different species of Aerodramus.5. Real time fluorescence quantitative PCR results showed that the size of PCR products detected by agarose electrophoresis were 270 bp and their sequences were from Aerodramus fuciphagus by sequencing analysis. The copy numbers of 5 positive standard samples and the corresponding CT value had a good linear relationship within the range of of 3.7×107 copies/μL to 3.7×103 copies/μL, the correlation coefficient R2=0.964, the slope was in the proper range (-3.38),the linear equation was CT=-3.38 (copy number)+16.304. Analysis of the specificity showed that amplification curves were observed when DNA from different sources and batches of EBN samples were used as the templates, while no amplification curve was observed when the mitochondrial DNA of human, rat, chicken, fish, and seeweed were used as the templates. These results suggested that the primers and probes we designed had good specificity for Cytb gene. When as low as 3.7×103copies/μL DNA sample was used as the template, we still observed amplification curve, indicating that our method had good sensitivity and could be used to detect low amount of EBN samples.6. Agrose electrophoresis analysis of PCR products amplified from EBN samples containing different proportions of pigskin showed an obvious band of 130 bp, which was confirmed as the sequence of pig by sequencing analysis. No PCR products were detected when DNA extracted from EBN samples without the addition of pigskin was used as the template. These data indicate that our method could effectively detect the pigskin added into EBN samples. ConclusionOur study showed that genetic origin of white nests is Aerodramus fuciphagus or its subspecies, and genetic origin of black nest is Aerodramus maximus or the subspecies Aerodramus maximuslowi. There are no correlation between genetic origin and EBN from different countries, with different colors, or with different shapes. However, genetic origin of most cave EBN is Aerodramus fuciphagus germani, while that of most breeding EBN is Aerodramus fuciphagus. We established DNA barcodes based on Cytb, ND2,COI and 12S rRNA sequence analysis for the rapid and accurate identification of the origin species of EBN samples. Due to the limitations on available EBN sample variants and gene sequence data deposited in Genbank, our method could not identify the subspecies of Aerodramus maximus. We developed real-time fluorescence quantitative PCR method that showed good specificity, repeatability and sensitivity to detect trace amounts of EBN, and our method could be used to rapidly and accurately identify fake EBN samples which contain pigskin components. |
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