Taqman rt pcr kit

Manufactured by Meridian Bioscience

Sourced in United States

The TaqMan RT-PCR kit is a laboratory equipment used for reverse transcription and real-time PCR analysis. It enables the detection and quantification of RNA targets in a sample.

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Lab products found in correlation

Ave buffer, by Qiagen (2 mentions) 7500 sequence detector, by Thermo Fisher Scientific (2 mentions) Stat 60 rna extraction reagent, by Tel-Test (2 mentions) Minelute virus spin kit, by Qiagen (1 mentions) Rna stat 60, by Qiagen (1 mentions)

4 protocols using taqman rt pcr kit

SARS-CoV-2 Detection via qRT-PCR Assay

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Viral RNA from oral swabs was isolated with the Qiagen MinElute virus spin kit (cat. no. 57704). Tissue RNA was extracted with RNA-STAT 60 (Tel-test”B″)/chloroform, precipitated and resuspended in AVE Buffer (Qiagen 1020953). The qRT-PCR assay utilizes reagents that bind to a conserved region of the N gene of SARS-CoV-2. The amount of RNA was determined by O.D. reading at 260, numbers of copies were calculated and 10⁸ copies per reaction were amplified. The mRNA PCR assay samples were amplified with TaqMan RT-PCR kit (Bioline cat# BIO-78005). Serial dilutions of a standard were included. Samples were amplified in an Applied Biosystems 7500 Sequence detector at 48 °C for 30 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, and 1 min at 55 °C.
Primers/probe sequences:
2019-nCoV_N1-F:5′-GAC CCC AAA ATC AGC GAA AT-3’.
2019-nCoV_N1-R: 5′-TCT GGT TAC TGC CAG TTG AAT CTG-3’.
2019-nCoV_N1-P: 5′-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3’.
The sgmRNA assays used the TaqMan RT-PCR kit (Bioline #BIO-78005 and was performed with an Applied Biosystems 7500 Sequence detector at 48 °C for 30 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, and 1 min at 55 °C.
Primers/probe sequences:
sg-N-F: 5′-CGATCTCTTGTAGATCTGTTCTC-3’.
sg-N-R: 5′-GGTGAACCAAGACGCAGTAT-3’.
Sg-N-P: 5′-6-FAM/TAACCAGAA/ZEN/TGGAGAACGCAGTGGG/3IABkFQ/

Hasanpourghadi M., Novikov M., Ambrose R., Chekaoui A., Newman D., Ding J., Giles-Davis W., Xiang Z., Zhou X.Y., Liu Q., Swagata K, & Ertl H.C. (2022). Heterologous chimpanzee adenovirus vector immunizations for SARS-CoV-2 spike and nucleocapsid protect hamsters against COVID-19. Microbes and Infection, 25(4), 105082.

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Quantifying SARS-CoV-2 RNA in Tissues

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The amounts of RNA copies per gram tissue were measured using a qRT-PCR assay, as described previously [35 (link)]. Briefly, viral RNA was extracted from the lung and nares collected on 7 dpi with RNA-STAT 60 (Tel-test “B”)/chloroform, precipitated and resuspended in AVE Buffer (Qiagen). To control the amplification reaction, RNA was isolated from the applicable virus stock using the same procedure. RT-PCR assays were performed using TaqMan RT-PCR kit (Bioline, BIO-78005) with primers and probe sequences, described previously [35 (link)]. The signal was compared to the known standard curve and calculated to give copies per gram (g). All samples were tested in triplicate.

Gu M., Torres J.L., Li Y., Van Ry A., Greenhouse J., Wallace S., Chiang C.I., Pessaint L., Jackson A.M., Porto M., Kar S., Li Y., Ward A.B, & Wang Y. (2021). One dose of COVID-19 nanoparticle vaccine REVC-128 protects against SARS-CoV-2 challenge at two weeks post-immunization. Emerging Microbes & Infections, 10(1), 2016-2029.

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SARS-CoV-2 Viral RNA Quantification from Lung Tissue

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Lungs RNA was extracted using RNA-STAT 60 extraction reagent (Tel-Test, Inc.) plus chloroform, precipitated, and re-suspended in RNAse-free water. Control RNA was isolated from SARS-CoV-2 viral stocks following the same procedure and quantified by OD260. These control stocks were serially diluted and OD260_nm values measured to generate a standard curve. RT-qPCR of the lung RNA was carried out using the following primers: 2019-nCoV_N1-F: 5'-GAC CCC AAA ATC AGC GAA AT-3'; 2019-nCoV_N1-R: 5'-TCT GGT TAC TGC CAG TTG AAT CTG-3'; and probe 2019-nCoV_N1-P: 5'-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3' (Integrated DNA Technologies) which were designed to bind to and amplify a conserved region of SARS-CoV-2 Nucleocapsid (N) RNA. Amplification was performed with an Applied Biosystems 7500 Sequence detector using the following program: 48°C for 30 min, 95°C for 10 min followed by 40 cycles of 95°C for 15 seconds, and 1 minat 55°C Reactions were carried out using a TaqMan RT-PCR kit (Meridian Bioscience, cat#BIO -78005) in 50 µL volume containing 5 µL of template, 2 µM of each primer and 2 µM of each probe. The number of viral RNA copies per mL was calculated by extrapolation from the standard curve, and values were then converted to the number of viral RNA copies per gram of lung tissue.

Brumeanu T.D., Vir P., Karim A.F., Kar S., Benetiene D., Lok M., Greenhouse J., Putmon-Taylor T., Kitajewski C., Chung K.K., Pratt K.P, & Casares S.A. (2022). Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2.HLA-DR4.Rag1KO.IL-2RγcKO.NOD) for COVID-19. Human Vaccines & Immunotherapeutics, 18(5), 2048622.

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SARS-CoV-2 RNA Quantification in Murine Lungs

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RNA from lungs of mice #M2–M4 and #F11-F13 was extracted using RNA-STAT 60 extraction reagent (Tel-Test, Inc., Friendswood, TX, USA) + chloroform, precipitated and resuspended in RNAse-free water. Control RNA was isolated from SARS-CoV-2 viral stocks following the same procedure and quantified by OD260. These control stocks were serially diluted and OD260 values measured to generate a standard curve. RT-qPCR of the lung RNA was carried out using the following primers : 2019-nCoV_N1-F :5’-GAC CCC AAA ATC AGC GAA AT-3’; 2019-nCoV_N1-R: 5’-TCT GGT TAC TGC CAG TTG AAT CTG-3’; and probe 2019-nCoV_N1-P: 5’-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1–3’ (Integrated DNA Technologies, Coralville, IA, USA) which were designed to bind to and amplify a conserved region of SARS-CoV-2 Nucleocapsid (N) RNA. Amplification was performed with an Applied Biosystems 7500 Sequence detector using the following program: 48°C for 30 minutes, 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds, and 1 minute at 55°C. Reactions were carried out using a TaqMan RT-PCR kit (Meridian Bioscience, Memphis, TN, USA) in 50 μL volume containing 5 μL of template, 2 μM of each primer and 2μM of each probe. The number of viral RNA copies per mL was calculated by extrapolation from the standard curve, and values were then converted to the number of viral RNA copies per gram of lung tissue.

Brumeanu T.D., Vir P., Karim A.F., Kar S., Benetiene D., Lok M., Greenhouse J., Putmon-Taylor T., Kitajewski C., Chung K.K., Pratt K.P, & Casares S.A. (2021). A Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO.IL-2Rγc KO. NOD) for COVID-19. bioRxiv.

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