Primestar max premix 2

The strains and the fruit bodies (part of) cultivated by the strain from the main producing areas of G. lingzhi in China were investigated. Fifteen fruit bodies and 22 strains of Lingzhi were collected (Additional file 1: Table S1). Strains were inoculated into the sterilized liquid fermentation medium (PD, autoclaved at 121 °C and 98 kPa for 20 min) in the shaker at 27 °C, 150 rpm for 9 days. Samples were then centrifuged at 8000 rpm to discard the medium, and mycelia were washed with pure water three times and prepared for use.
The TaKaRa MiniBEST Plant Genomic DNA Extraction Kit and PCR mix [Prime STAR Max Premix (2 ×) bought from Takara Biomedical Technology (Beijing) Co., Ltd.] were used for extracting total genomic DNA and PCR of 22 strains, respectively. The ITS primers and PCR protocol were applied as previously described (White et al. 1990 ). The primers synthesized and PCR products sequencing were obtained from Beijing Genomics Institute (BGI).

Complementary DNA was synthesized from total RNA (1 µg) using a Prime-Script RT PCR Kit (Takara Bio Inc.), according to the manufacturer’s instructions. The confirmation of the quantification of the splicing variant was performed as previously described [16 (link)]. In detail, total RNA was reverse transcribed to cDNA, and PCR amplification was performed using PrimeSTAR Max Premix (2) (Takara Bio Inc.), with the primers shown in Supplementary Material, Table S2. Each splicing variant of the PCR product was shown using an Agilent 2100 Bioanalyzer performed with a DNA 1000 LabChip Kit and High sensitivity LabChip Kit (Agilent Technologies Inc.). For semi-quantification, the proportions of splicing variant were calculated using the following formula: proportion = amount of the one product/ (amount of the one product + amount of the other product).

Seventeen children (aged 3–9 years) participated in this study (ethics committee approval no. EC20-28; Nihon University School of Dentistry at Matsudo, Chiba, Japan). The decayed, missing, and filled tooth indices of these patients ranged from 4 to 16. Plaque was collected from the tooth surface using a sterile excavator, suspended in 1 mL of 0.9% (w/v) NaCl solution, applied to mitis–salivarius agar, and incubated at 37 °C for 48 h. Rough colonies, characteristic of S. mutans, were isolated. Species in the genus were identified based on the presence or absence of a portion of the htrA gene encoding a surface protease and an intergenic region following polymerase chain reaction (PCR) amplification, as reported by Chen et al. [10 (link)]. Genomic DNA was extracted from the culture medium using ISOPLANT (Nippon Gene Co., Ltd., Tokyo, Japan) following the manufacturer’s protocols. The PCR mixture was prepared by mixing 6 µL PrimeSTAR Max Premix (2×) (Takara Bio Inc., Shiga, Japan), 10 ng genomic DNA, forward (5′-TCGCGAAAAAGATAAACAAACA-3′) and reverse (5′-GCCCCTTCACAGTTGGTTAG-3′) primers (final concentration of 0.2 µM each), and PCR-grade water. The PCR conditions were as follows: 30 cycles at 98 °C for 10 s, 55 °C for 10 s, and 72 °C for 6 s. The PCR products were subjected to MultiNA capillary electrophoresis (MCE-202; Shimadzu Corporation, Kyoto, Japan) to confirm amplification.