TABLE 1.
Species-specific primers used in this study
| Primer name | Primer sequence (5′–3′) | Species | GenBank ID | Standard curvea | Amplicon length | Reference or source |
|---|---|---|---|---|---|---|
| PLace F | AAAAAGCAGAGTGGAGAAAATACT | L. acetotolerans | BAQ57713.1 | y = −3.65x + 34.67; R2 = 0.99; 10.7–1.07 × 108 (copies/reaction) | 119 bp | This study |
| PLace R | CCAATAAAAAGAGCAACAGCA | |||||
| PLjin F | CGCACTCCCGTAGATGATTTTGA | L. jinshani | KU674948.1 | y = −3.54x + 33.85; R2 = 0.99; 17.8–17.8 × 108 (copies/reaction) | 445 bp | (21) |
| PLjin R | TCACTACCAAGCCATTTCCTAC | |||||
| PPpen F | CTATTGACTTGGTCGTTATTGATTCC | P. pentosaceus | WP_002833728 | y = −3.14x + 33.33; R2 = 0.99; 28.8–2.88 × 108 (copies/reaction) | 72 bp | (41) |
| PPpen R | CCCCCATCTCTCCATCAATTT | |||||
| PWpar F | CTAGAGGCGGCGAAGTCAGT | W. paramesenteroides | ATF40921.1 | y = −3.44x + 36.79; R2 = 0.99; 17–1.70 × 108 (copies/reaction) | 101 bp | This study |
| PWpar R | CTATTCGCGTCGCCAACCAT | |||||
| PBcoa F | CTCACGGAAGAGCAAGCTTG | B. coagulans | QAU28402.1 | y = −3.23x + 36.74; R2 = 0.99; 10.7–1.07 × 108 (copies/reaction) | 293 bp | (42) |
| PBcoa R | GTTTCTGAAATGTATGCACG |
x is the Ct value; y is the log10 of the number of gene copies per reaction. The amplification conditions of PLace, PPpen, PWpar, and PBcoa were 95°C for 5 min, followed by 40 cycles of 95°C for 5 s and 60°C for 20 s. The amplification conditions of PLjin were 98°C for 1 min, followed by 40 cycles of 98°C for 10 s, 55°C for 30 s and 72°C for 25 s. Species-specific gene (GenBank BAQ57713.1) for L. acetotolerans was screened from genome sequence GenBank AP014808.1. Species-specific gene (GenBank ATF40921.1) for W. paramesenteroides was screened from genome sequence GenBank: CP023501.1. The species-specific primers PLace and PWpar were designed using Batchprimer 3 (40) with the following parameters: amplicon size, 100 to 300 bp; amplicon GC content, 40 to 60% (optimal 50%); primer length, 18 to 28 nt (optimal 20 nt); and melting temperature (Tm) 57 to 63°C (optimal 60°C, maximum difference per primer pair <3°C).