Quantitect multiplex pcr kit

RNA was isolated 472 hr post-transfection and reversely transcribed using SuperScript VILO kit (Thermo Scientific). All qPCR analyses were performed with the QuantiTect Multiplex PCR kit (Qiagen, Hilden, Germany) on a StepOne Real-Time PCR Sytem (Applied Biosytems) with a total of 45 cycles. Constitutive maintenance gene 18S rRNA (Applied Biosystems, Quencher VIC/MGB, 4319413E) and human WRN (Applied Biosystems, Quencher FAM/MGB-NFQ, 4331182), human MLH1 (Applied Biosystems, Quencher FAM/MGB-NFQ, 4453320) and human MSH3 (Applied Biosystems, Quencher FAM/MGB-NFQ, 4448892) TaqMan probes were used. WRN expression was normalized to 18S rRNA expression levels and is indicated relative to the NTC control.

Real-time PCR for detection of CMV was performed with the QuantiTect Multiplex PCR kit (Qiagen), and amplification was conducted using the QuantStudio^TM3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) as previously described^{44 (link)}. To confirm the presence of viruses detected by NGS, reverse transcription PCR (RT-PCR) was performed with the Cycleave PCR respiratory virus detection kit (Takara Bio, Kusatsu, Japan), and amplification was conducted using the QuantStudio^TM3 Real-Time PCR System in accordance with the manufacturer’s instructions. Real-time PCR for S. epidermidis and E. faecalis was performed by a commercial laboratory (TechnoSuruga Laboratory, Shizuoka, Japan) using Rotor-Gene (Qiagen). Information about the primers and probes used for PCR is shown in Supplementary Table 2^{44 (link)–49 (link)}.

Based on VNTR 3, a TaqMan qPCR was established using two different probes. One probe covered the sequences shown in the VNTR 3 of the European KHV lineage and the other probe covered sequences of the same VNTR, but only the Asian KHV lineage was selected (Table 3). This was combined with sequence data obtained with forward and reverse primers (Table 2). For TaqMan qPCR, the QuantiTect Multiplex PCR Kit (Qiagen) was used following the manufacturer's instructions. Initially a denaturation step at 95°C for 15 min was started followed by 42 cycles with thermal profile: 95°C for 1 min, 60°C for 30 s and 72°C for 30 s. All samples were tested in the presence of three negative controls (water). The threshold was set to C_q = 39 (~1–5 particles/ml, Bergmann et al., 2010 (link)) for discrimination between positive and false positive samples.

DNA was extracted using the prepGEM™ Insect kit DNA extraction kit (ZyGEM Corporation Ltd) following the manufacturers’ instructions. Samples were amplified at 13 microsatellite loci reported in Santana et al. (2009), using the QuantiTect^® Multiplex PCR Kit (Qiagen) and primers from Inqaba Biotec (Pretoria) (Santana et al., 2009). Amplification was performed using the manufacturer's instructions but modified such that all reactions were scaled down to a total volume of 8.2 µl. Cycling conditions were 95°C for 15 min followed by fifty cycles of 94°C for 1 min and 60°C for 1.5 min.
Amplified products were visualized using agarose gel electrophoresis on a 2% agarose gel. PCR products were loaded with 30× GelRed (Biotium). Visualized PCR products were analyzed by electrophoresis in an ABI PRISM 3100 Automated DNA Sequencer (Applied Biosystems). The data collected were analyzed using GeneMapper software (version 3.0; Applied Biosystems) for genotyping of all samples at the 13 loci. All allele calls made by the software were checked manually, and in cases where complete genotypes were not obtained, samples were excluded from further analyses.

Viral RNA for testing by rPCR assay was extracted by a MagMAX CORE Nucleic Acid Purification Kit (Thermo Fisher Scientific) using 200 µL of UTM mixed with clinical specimens according to the manufacturer's instructions, with an elution of 50 µL. cDNA was synthesized with a random hexamer primer using the PrimeScript RT reagent kit (Takara Bio, Shiga, Japan) and 10 µL viral RNA. Multiplex rPCR was conducted using the QuantiTect multiplex PCR kit (Qiagen, Hilden, Germany) for detecting IAV and IBV, RSV A and B, and hMPV as described previously^{21 (link)}. The rPCR was performed in a 20 µL reaction containing 5 µL cDNA as the template and using the LightCycler 480 II (Roche, Basel, Switzerland). The RSV A and RSV B results were not separate, but were judged comprehensively and used as RSV results in this study.

A total of 18 patients with pediatric acute encephalitis/encephalopathy of unknown etiology were enrolled in this study (Table 1). Acute encephalopathy/encephalitis was defined as occurring in patients with a depressed or altered level of consciousness lasting more than 24 hours and one or more of the following: fever (>38 °C) during the presenting illness; seizure(s) and/or focal neurological findings; CSF pleocytosis; abnormal results of an electroencephalogram; abnormal results of neuroimaging1 (link). Cultures of blood and CSF for bacteria, rapid influenza test, and PCR for HSV, HHV-6, and HHV-7 were negative in all patients. RT-PCR for enterovirus was not performed at enrollment. Serum and CSF samples were obtained in the acute phase (within 2 days of the onset of neurologic involvement) and stored at −30 °C until use. Clinical samples obtained from patients with a defined diagnosis of viral infection such as adenovirus hepatitis, HSV encephalitis, hepatitis C virus (HCV), or HHV-6 encephalopathy were used to validate the NGS-based approach to detect virus-derived sequences. Real-time PCR of HHV-6 was performed with a QuantiTect multiplex PCR kit (Qiagen, Hilden, Germany) as described previously21 (link), and primers and probes are shown in Table 2.