A 7.5-μL duplexed PCR probe assay was composed of 1X qScript XLT 1-Step RT-qPCR ToughMix (QuantaBio), 0.1 U/μL SpeedSTAR HS DNA polymerase (Takara Bio), 0.1 U/μL AccuStart II Taq DNA polymerase (QuantaBio), 1 mg/mL BSA (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich) and primer-probe pairs. For duplexed assay for N1 and control RNA, the assay contains 1 μM each N1 primer, 0.45 μM each Luciferase primer, 1 μM N1 probe and 0.25 μM Luciferase probe. For duplexed assay for influenza A and influenza B, the assay contains 0.5 μM each influenza A primer, 1 μM each influenza B primer, 0.25 μM influenza A probe and 0.5 μM influenza B probe. For duplexed assay of SARS-CoV-2 variant detection, the assay contains 0.67 μM each Yale Spike Δ69–70 primer, 0.3 μM each Yale ORF1a Δ3675–3677 primer, 0.2 μM Yale Spike Δ69–70 probe and 0.2 μM ORF1a Δ3675–3677 probe. All oligonucleotides, including primers and fluorescently labeled DNA probe (sequences in Supplementary Table 4 ) were purchased from Integrated DNA Technologies.
Speedstar hs dna polymerase
Manufactured by Takara Bio
Sourced in Japan, China
SpeedSTAR HS DNA polymerase is a high-speed, high-fidelity DNA polymerase designed for efficient and accurate DNA amplification. It exhibits rapid DNA extension, enabling faster PCR run times. The enzyme possesses 3' to 5' exonuclease proofreading activity to ensure high-fidelity DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
Accustart 2 taq dna polymerase, by Quantabio (1 mentions)
Qscript xlt 1 step rt qpcr toughmix, by Quantabio (1 mentions)
Gel documentation system, by Syngene (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Nahco3, by Thermo Fisher Scientific (1 mentions)
Nz microbiological bsa, by MP Biomedicals (1 mentions)
Dntp mixture, by Takara Bio (1 mentions)
Fast buffer 1, by Takara Bio (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Tianscript first strand cdna synthesis kit, by Tiangen Biotech (1 mentions)
Pmd19 t vector, by Takara Bio (1 mentions)
Solution 1, by Takara Bio (1 mentions)
Dneasy tissue kit, by Qiagen (1 mentions)
Primestar gxl dna polymerase, by Takara Bio (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
High pure pcr cleanup micro kit, by Roche (1 mentions)
One step primescript rt pcr kit, by Takara Bio (1 mentions)
9 protocols using speedstar hs dna polymerase
1
Multiplex SARS-CoV-2 Variant Detection Assay
2
Molecular Detection of Theileria annulata
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PCR was used to amplify 125 bp fragment of T. annulata 18S rRNA gene [Genbank:EU083801.1] with species-specific primers, Forward (5′- ACGACTCCTTCAGCACCTTG-3′) and Reverse (5′-AAATTAAGCCGCAGCTCCAC-3′). PCR reactions were performed using Speed Star HS DNA polymerase (Takara) in an automatic DNA thermocycler (Biorad-T100 Thermal cycler). Reactions were performed in 25 μl volumes with 100 ng of purified genomic DNA. PCR conditions consisted of a first denaturation step of 1 min at 95 °C, followed by a second step of 35 cycles of 10 s at 95 °C, 20s at 61 °C and 10 s at 72 °C, and a final extension step of 1 min at 72 °C. DNA extracted from in vitro cultured T. annulata parasite was used as positive whereas distilled water and T. oreintalis positive DNA sample were used as negative controls, respectively in all PCR reactions. PCR products were separated by 2 % agarose gel electrophoresis and stained with ethidium bromide to assess the presence of specific bands indicative of T. annulata . Gels were photographed under gel documentation system (Syngene) and the size of each PCR product determined by reference to a DNA ladder (NEB).
3
Cultivation and Genotyping of Malaria Parasites
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P. falciparum laboratory lines were cultivated at 37 oC under 5% O2, 5% CO2, 90% N2 in O+ human erythrocytes obtained from Interstate Blood Bank (Memphis, TN) at 5% hematocrit in RPMI 1640 medium (KD Medical) supplemented with 25 mM HEPES, 50 μg/mL hypoxanthine, 0.5% NZ Microbiological BSA (MP Biomedicals), gentamicin and 28.6 mM NaHCO3 (Gibco). A modified medium, PGIM [14 (link)], follows the RPMI 1640 medium with supplements but has reduced concentrations of isoleucine and hypoxanthine (11.4 μM and 3.01 μM, respectively). Primers specific to 5’ and 3’ UTR regions of clag3.1 and clag3.2 were used to detect the number and possible combinations of clag3 alleles in the GB4 and 7G8 parental lines (S2 Table ; SpeedSTAR HS DNA polymerase, Takara).
4
Rapid Real-time PCR for HSV and VZV
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Quantitative real-time PCR was performed using a microfluidic real-time PCR system (GeneSoC®, Kyorin, Tokyo, Japan). Gene SoC® can show three colors; therefore, simultaneous measurements of HSV DNA (Cy5) and VZV DNA (FAM) were performed in this study. Since the time required for real-time PCR was approximately 12 min, the HSV and VZV DNA test results were available in 40 min following the collection of tears.
The PCR mix contained cold lysis buffer including DNA extracts 200 μL, HSV-1 primer & probe [Probe (Cy5): TAG TGG GCC TCC ATG GG, HSV1F: GGG CCG TGA TTT TGT TTG TC, HSV1R: CCG CCA AGG CAT ATT TGC] 2.5 μL, HHV3 TaqMan probe (FAM) (TaqMan gene expression assay, Life Technologies Japan, Carlsbad, Tokyo, Japan) 1.0 μL, 10 × FAST Buffer I (Takara Bio Inc, Shiga, Japan) 2.0 μL, dNTP Mixture (Takara Bio) 1.6 μL, SpeedSTAR™ HS DNA Polymerase (Takara Bio) 0.4 μL, and distilled water 3.9 μL.
The PCR mix contained cold lysis buffer including DNA extracts 200 μL, HSV-1 primer & probe [Probe (Cy5): TAG TGG GCC TCC ATG GG, HSV1F: GGG CCG TGA TTT TGT TTG TC, HSV1R: CCG CCA AGG CAT ATT TGC] 2.5 μL, HHV3 TaqMan probe (FAM) (TaqMan gene expression assay, Life Technologies Japan, Carlsbad, Tokyo, Japan) 1.0 μL, 10 × FAST Buffer I (Takara Bio Inc, Shiga, Japan) 2.0 μL, dNTP Mixture (Takara Bio) 1.6 μL, SpeedSTAR™ HS DNA Polymerase (Takara Bio) 0.4 μL, and distilled water 3.9 μL.
5
Isolation and Cloning of Full-Length cDNA
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Total RNA was isolated from Chinese jujube (Z. jujuba), apple (Malus × domestica), peach (P. persica) and pear (Pyrus × bretschneideri) by using an RNAprep Pure Plant Kit (Polysaccharides & Polyphenolics-rich; Tiangen, China) in accordance with the manufacturer’s instructions. The gene-specific primers were designed according to the accession numbers XM_016033463.2, XM_008345245.2, XM_007218867.2 and XM_01864 2751.1 (Table S2 ). We used a TIANScript First Strand cDNA Synthesis Kit (Tiangen, China) to synthesize first-strand complementary DNA (cDNA). Full-length cDNA was obtained by performing PCR in a 50 μL volume that comprised 0.25 μL of SpeedSTAR HS DNA Polymerase (TaKaRa, China), 4 μL of a dNTP mixture (2.5 mM), each primer at 10 μM, 2 μL of cDNA, and 5 μL of 10× Fast Buffer I (Mg2+ plus), with ddH2O added to reach final volume of 50 μL. The PCR amplification procedure was as follows: 5 min at 95 °C; 35 cycles of 8 s at 98 °C, 20 s at 60 °C, and 20 s at 72 °C; and 10 min at 72 °C. The PCR product was preserved at 4 °C. All target fragments were cloned into a pMD19-T vector (TaKaRa, China) using Solution I (TaKaRa, China) and then transformed to DH5а (Tiangen, China).
6
DNA Extraction and Sequencing Protocol for Lepidodactylus lugubris
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Lepidodactylus lugubris sample was collected at Chichi-jima Island of the Bonin Islands, Japan. Other animal samples of either dead or live individuals were obtained from local shops or animal dealers in Japan. All experiments in which live animals are handled were conducted carefully under the guideline of the Animal Experiment Committee of Nagoya City University with permission (No. H21N-02).
A small amount of tissue from the tail muscle was used for crude DNA extraction with a DNeasy Tissue Kit (Qiagen). PCR amplifications were conducted with SpeedSTAR HS DNA polymerase (Takara) or PrimeSTAR GXL DNA polymerase (Takara) according to the manufacturer’s instructions. The former was routinely used for <2 kbp amplifications and the latter was selected for longer amplifications. Short amplified products were purified with a High Pure PCR Cleanup Micro Kit (Roche), followed by the dye termination sequencing reaction using a BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies). The resultant reaction mixture was ethanol precipitated and applied to the 8-capillary 3500 Genetic Analyzer (Life Technologies) in the standard run mode.
A small amount of tissue from the tail muscle was used for crude DNA extraction with a DNeasy Tissue Kit (Qiagen). PCR amplifications were conducted with SpeedSTAR HS DNA polymerase (Takara) or PrimeSTAR GXL DNA polymerase (Takara) according to the manufacturer’s instructions. The former was routinely used for <2 kbp amplifications and the latter was selected for longer amplifications. Short amplified products were purified with a High Pure PCR Cleanup Micro Kit (Roche), followed by the dye termination sequencing reaction using a BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies). The resultant reaction mixture was ethanol precipitated and applied to the 8-capillary 3500 Genetic Analyzer (Life Technologies) in the standard run mode.
7
Chromosomal Modification of P. furiosus with Codh Gene Cluster
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The 22.2 kb BAC vector, pGL058 (Basen et al., 2014 (link)), containing the Codh gene cluster expression construct for homologous recombination into the P. furiosus chromosome at genome region five between convergent genes PF1232 and PF1233 was linearized using the unique XhoI restriction site (within repE in the BAC vector backbone) and used to transform P. furiosus COM1 according to previously published methods (Lipscomb et al., 2011 (link)). Transformant colonies were cultured in defined cellobiose (DC) medium (Lipscomb et al., 2011 (link)), and gDNA was isolated using the ZymoBeadTM Genomic DNA Kit (Zymo Research) for PCR screening. PCR screens were performed using the SpeedSTAR HS DNA polymerase (Takara) for amplification of the ∼16 kb product with primers ∼100 b outside the chromosomal homologous flanking regions. PCR-verified isolates were further purified twice on solid DC medium prior to final PCR screening and saving of glycerol stocks. One of the purified pGL058 transformants, designated MW191, was selected for phenotypic analyses. This strain is referred to herein as the Codh strain.
8
GeneSoC Thermal Cycling for Rapid RT-PCR
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GeneSoC® uses heaters to control the temperature for reverse transcription, denaturation, annealing, and extension processes, with thermal cycling performed by sending the PCR solution to and from each heater. Accordingly, this microfluidic thermal cycling through the roundtrip transport of preheated denaturation, annealing, and extension processes drastically reduces the time required for temperature changes in the sample solution. The GeneSoC® device is composed of a main unit and a detection unit. The system is expandable to run up to four detection units on a single main unit. All components of the system are integrally controlled [5 , 6 (link)]. The reaction kit and polymerase used for this study were the One Step Prime Script RT-PCR kit (Takara Bio Inc., Shiga, Japan) and Speed STAR HS DNA polymerase (Takara Bio Inc.), as described in Table 1A and 1B . Reaction reagent (15 μL) was mixed with template RNA (5 μL), and the solution was dispensed onto a reaction panel chip customized for GeneSoC®. Primers and probes were targeted to E genes (hereinafter, E primer) and RdRp genes (hereinafter, RdRp primer). The primer and probe arrangement (5′ to 3′) (Table 2 ) [7 ] and PCR conditions (Table 3 ) were as shown.
9
SARS-CoV-2 Detection Using Cas12a-Based Assay
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Sangon Biotech. (Shanghai, China) synthesized all primers, single stranded DNA (ssDNA) reporters and crRNAs. DNase I, EngenLba Cas12a and kits used for HiScribe T7 High Yield RNA Synthesis were bought from New England Biolabs (Ipswich, MA, USA). SpeedSTAR HS DNA Polymerase, PrimeScript RT Master Mix and RNase inhibitor were offered by Takara Bio Inc. (Dalian, China). QIAGEN OneStep RT-PCR kit was bought from Qiagen (Frederick, MD, USA). EvaGreen dye was purchased from Biutium (Shanghai, China). The ddPCRsupermix for probes (no dUTP) was purchased from Bio-Rad Lab. Inc. (CA, USA). Tiangen Biotechnology Co., Ltd. (Beijing, China) provided the DNA purification kit and RNA clean kits. Magnetic beads based nucleic acid extraction kit was kindly provided by Lingjun Biotechnology Co., Ltd. (Shanghai, China). BGI (Beijing, China) offered the pUC57 plasmids inserted the SARS-CoV-2 gene sequences. Sangon Biotech. (Shanghai, China) also sold us the plasmids inserted gene sequences from bat SARS-like coronavirus, SARS-CoV and one human coronaviruses (HKU1). The First People's Hospital of Yuhang district (Hangzhou, China) offered the 20 clinical samples with SARS-CoV-2 infection and 20 control samples. Scientific Research Ethics Review Committee in the hospital offered samples approved our study. Throat swap samples were collected with written or oral informed consents form patients.
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