Minibest plasmid purification kit

In order to establish the quantitative assay, plasmids containing the target sequences of A.actinomycetemcomitans 16S rDNA and cdt gene were cloned using the pMD 19 T-Vector (Takara, Japan). PCR products for A.actinomycetemcomitans 16S rDNA and cdt gene were inserted into plasmid vectors respectively, and the recombinant vectors were transformed into E. coli. Then, the plasmids were purified with Mini BEST Plasmid Purification Kit (Takara, Japan). The purified plasmids were quantified by spectrophotometry. Standard curves were constructed by using serial diluted purified plasmids with predetermined concentrations on the basis of the linear relationship between the Ct and the logarithm of the starting gene amount. Sensitivity of the developed real-time PCR assay was evaluated by using 10⁷–10° plasmid copies of A. actinomycetemcomitans and cdtB gene (data not shown), limit of approximately 10 cells was established in the PCR reaction mixture.

The cDNA sequence of CgGLS-1 containing glutaminase domain was cloned into pET-30a vector (Primers were shown in Table 1). Restriction enzymes BamH I and Hind III were used to construct recombinant plasmids. The recombinant plasmid was isolated by MiniBEST plasmid purification kit (Takara, Japan) and then transferred into E. coli Transetta (DE3) (Transgen, China). Isopropyl β-D-Thiogalactoside (IPTG) (1 mmol/L) was used to induce the expression of recombinant protein, and the recombinant protein CgGLS-1 (designated rCgGLS-1) was purified by a Ni²⁺chelating Separate column (Sangon Biotech, China). The purity of obtained rCgGLS-1 was evaluated by SDS–polyacrylamide gel electrophoresis. An enhanced BCA protein assay kit (Beyotime, China) was used to quantify the content of rCgGLS-1^{46 (link)}. The purified protein was stored at −80 °C before use.

The partial gene of 23S rRNA was amplified by PCR using primers indicated above. Briefly, 12.5 μL of 2x PCR premix solution (Premix Taq™ Version 2.0, TaKaRa, Dalian, China), 1.5 μL of each primer (10 nM), 2 μL of genomic DNA (1 × 10⁸ copies/μL), and 7.5 μL of dH2O were mixed together to initiate the reaction. The reaction was conducted as described by the manufacturer's instruction. Plasmid pUC19 was purified from E. coli cells using a TaKaRa MiniBEST Plasmid Purification Kit. Both the amplified gene and plasmid were digested using BamH I and EcoR I restriction enzymes and linked using a Takara DNA Ligation Kit. Competent E. coli cells were transformed with ligation product, scraped to solid LB medium with ampicillin, and incubated overnight at 37°C. Recombinant plasmid 23SrRNA-pUC19 from positive bacterial colony was purified and digested using both BamH I and EcoR I, and the product was analyzed by agarose gel electrophoresis to confirm the target gene was linked. The concentration of the recombinant plasmid was measured using Nanodrop 2000, and the copies were calculated according to Avogadro constant.

In order to obtain the best reaction parameters, the multiplex PCR was optimized by varying single parameters while other parameters were maintained.The optimization was performed in a 50 μL PCR reaction mixture as follows: 10 × PCR buffer 5.0 μL, 10 mM dNTPs 2–4 μL, each 10 μM primer (Table 1) 0.5–1 μL, Taq DNA polymerase (5 U/μL) (TaKaRa, Dalian, China) 0.5–1 μL, the DNA template 3.0–5.0 μL(100 copies/μL), and added distilled water to 50 μL.
The amplifications were performed under the following conditions in a thermal cycler (Bio-Rad, Hercules, CA, USA). After 5 min initial denaturation at95 °C, 35 cycles were conducted at 94 °C for 40 s, 52–58 °C for 40 s and 72 °C for 50–70 s, followed by a 10-min final extension at 72 °C. The PCR products were detected according to our previous study [20 (link)].The specific viral target fragments were clonedinto the plasmid pMD18-T (TaKaRa, Dalian, China).
Plasmids containing the PCV1, PCV2 or PCV3 gene were purified using a MiniBEST Plasmid Purification Kit (TaKaRa, Dalian, China). Each plasmid sample concentration was determined by measuring the absorbance at 260 nm using a Eppendorf BioSpectrometer (Eppendorf, Hamburg, Germany), and each cloned gene copy number was quantified as previously described [21 (link)]. The standard PCV1, PCV2 and PCV3 DNA samples were ten-fold diluted (from 10⁷ to 10⁻²copies/μL) and stored at − 80 °C until use.