To investigate the possible aetiological agents associated with this disease, we collected bronchoalveolar lavage fluid (BALF) and performed deep meta-transcriptomic sequencing. The clinical specimen was handled in a biosafety level 3 laboratory at Shanghai Public Health Clinical Center. Total RNA was extracted from 200 μl of BALF and a meta-transcriptomic library was constructed for pair-end (150-bp reads) sequencing using an Illumina MiniSeq as previously described4,6,7,8. In total, we generated 56,565,928 sequence reads that were de novo-assembled and screened for potential aetiological agents. Of the 384,096 contigs assembled by Megahit9, the longest (30,474 nucleotides (nt)) had a high abundance and was closely related to a bat SARS-like coronavirus (CoV) isolate—bat SL-CoVZC45 (GenBank accession number MG772933)—that had previously been sampled in China, with a nucleotide identity of 89.1% (Supplementary Tables 1, 2). The genome sequence of this virus, as well as its termini, were determined and confirmed by reverse-transcription PCR (RT–PCR)10 and 5′/3′ rapid amplification of cDNA ends (RACE), respectively. This virus strain was designated as WH-Human 1 coronavirus (WHCV) (and has also been referred to as ‘2019-nCoV’) and its whole genome sequence (29,903 nt) has been assigned GenBank accession number MN908947.
Y ahora el procedimiento que se usa habitualmente desde hace varios años para definir a un virus como aislado:
Science Magazine - 15 January 2016 - page24
Te lo lees, es sencillo, luego miras la página 226 con más atención y aprendes que AHORA lo que se hace es secuenciar el genoma junto con el cultivo a partir de inoculaciones preparadas.
Tenéis un concepto erróneo de lo que en el mundo científico se entiende comúnmente por "aislamiento"
Los científicos llaman comúnmente aislamiento de un virus al cultivo de un virus en un medio celular artificial para observar sus efectos citopáticos.
Por eso lo que ves en todos esos papers son fotos del cultivo celular. Ese es el aislamiento del virus para observar efectos citopáticos.
Vemos el procedimiento de los Koreanos cogen exudado de la faringe, lo.meten en medio de cultivo a temperatura. Lo inoculan en células Vero como medio de cultivo. Al cabo del tiempo a las células les pasan cosas, la cortan con un microtomo y la meten al microscopio electrónico. Ven unas bolitas extrañas con unas características. Como secuencian el ADN:
For whole genome sequencing of the virus isolate (BetaCoV/Korea/SNU01/2020), culture supernatant of Vero cells infected was used for RNA extraction. RNA was extracted by using QIAamp viral RNA mini kit (QIAGEN, Valencia, CA, USA), according to the manufacturer's instructions. Ahí vemos el kit que usan.
Esta Magufa dice que hay que hacer centrifugación para homogenizar partículas por densidad (que es lo que se hace con eso).
El kit en cuestión en su web dice textualmente:
Optimized buffers and enzymes lyse samples, stabilize nucleic acids, and enhance selective DNA adsorption to the QIAamp membrane. Alcohol is added and lysates loaded onto the QIAamp spin column. Wash buffers are used to remove impurities and pure, ready-to-use DNA is then eluted in water or low-salt buffer.
No mechanical homogenization is necessary as the tissues are lysed enzymatically, and the convenient spin-column procedure means that hands-on preparation time is only 20 minutes (lysis times differ according to the sample source). Samples can be processed using either a microcentrifuge or, if blood or other body fluids are being processed, using the QIAvac 24 Plus or QIAvac 6S vacuum manifold. In addition, the rigorous lysis procedure employed makes the QIAamp DNA Mini Kit ideal for purification of genomic DNA from bacteria or parasites. To further reduce hands-on time, genomic DNA purification may be automated on the QIAcube.
Anda a mamarla soplapollas. Oda ya os ha puesto a caldo pero os hace falta más. Os creéis antes a esta que leer el artículo y entender lo que se ha hecho. Y el virólogo que se lo haga mirar que está algo desactualizado.
Si no te gusta, no es mi problema, pero deja de decir que el sars cov 2 no ha sido aislado, porque te estás poniendo en evidencia.
BLA BLA BLA... NADA DE AISLAMIENTO.Virus isolation, cell infection, electron microscopy and neutralization assay
The following cell lines were used for virus isolation in this study: Vero E6 and Huh7 cells...
Cultured cell monolayers were maintained in their respective medium...
Vero E6 cells were infected with the new virus ...
NADA DE AISLAMIENTO. USARON EL CULTIVO TAL CUAL, PARA EXTRAER EL RNARNA extraction and PCR
Whenever commercial kits were used, the manufacturer’s instructions were followed without modification.
RNA was extracted from 200 μl of samples with the High Pure Viral RNA kit (Roche).
RNA was eluted in 50 μl of elution buffer and used as the template for RT–PCR.
NADA DE AISLAMIENTO. USARON EL CULTIVO TAL CUAL, PARA EXTRAER EL RNAFirst, viral RNAs were extracted directly from clinical samples with the QIAamp Viral RNA Mini Kit, and then used to synthesise...
NADA DE AISLAMIENTO. NADA. LO MISMO, SECUENCIARON EL CULTIVO TAL CUAL ("culture supernatants"). PARA MÁS INRI, EL CULTIVO PRESENTABA CONTAMINACIÓN DE MYCOPLASMAS.The virus titers in culture supernatants of the five cases at 3 d p.i. were 4.6 × 106 to 6.8 × 107 median tissue culture infectious dose (TCID50) per mL (Table 1).
Next-generation sequencing (NGS) of case Wk-521 detected the nearly full-length genome sequence from SARS-CoV-2 with >99.9% homology (1, 2) (GISAID database ID EPI_ISL_408667).
Unexpectedly, the NGS data showed contaminated mycoplasma sequences (Mycoplasma hyorhinis and Mycoplasma arginini) from VeroE6/TMPRSS2 cells.
NADA DE AISLAMIENTO. NADA. LO MISMO, SECUENCIARON EL CULTIVO TAL CUAL (el lisado de todo lo que hubiera allí).Cell culture, limiting dilution, and isolation:
Vero CCL-81 cells were used for isolation and initial passage. Vero E6, Vero CCL-81, HUH 7.0,
293T, A549, and EFKB3 cells were cultured in Dulbecco’s minimal essential medium (DMEM)
supplemented with heat inactivated fetal bovine serum (5 or 10%) and anti/anti antibiotic
(GIBCO). Both NP an OP swabs were used for virus isolation. For the isolation, limiting
dilution, and passage 1 of the virus, 50 µl serum free DMEM was pipetted into columns 2-12 of
a 96-well tissue culture plate. One-hundred µl clinical specimens were pipetted into column 1,
and then serially diluted 2-fold across the plate. Vero cells were trypsinized and resuspended in
DMEM + 10% FBS + 2X Penicillin-Streptomycin + 2X antibiotic – antimycotic + 2 X
amphotericin B at 2.5 x 105 cells / ml. One hundred µl of cell suspension were added directly to
the clinical specimen dilutions and mixed gently by pipetting. The inoculated cultures were
grown in a humidified 37°C incubator with 5% CO2 and observed for cytopathic effect (CPE)
daily. Standard plaque assays were used for SARS-CoV-2 based on both SARS-CoV and
MERS-CoV protocols (16, 17).
When CPE were observed, the cell monolayers were scraped with the back of a pipette tip. Fifty
µl of the viral lysate were used for total nucleic acid extraction for confirmatory testing and
sequencing. Fifty µl of virus lysate was used to inoculate a well of a 90% confluent 24-well
NADA DE AISLAMIENTO. NADA. LO MISMO, SECUENCIARON EL CULTIVO TAL CUAL (culture supernatant). Aparece nuestro "amigo" el QIAamp viral RNA mini kit!!!!For whole genome sequencing of the virus isolate (BetaCoV/Korea/SNU01/2020), culture supernatant of Vero cells infected was used for RNA extraction.
RNA was extracted by using QIAamp viral RNA mini kit (QIAGEN, Valencia, CA, USA), according to the manufacturer's instructions.
NADA DE AISLAMIENTO. NADA. LO MISMO, SECUENCIARON EL CULTIVO TAL CUAL (cultured cell medium). Aparece nuestro "amigo" el QIAamp viral RNA mini kit!!!!RNA was extracted from clinical samples with a QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions
Using reverse transcriptase, cDNA was synthesized from RNA extracted from the cultured cell medium in which the virus was replicated.
NADA DE AISLAMIENTO. NADA. LO MISMO, SECUENCIARON EL CULTIVO TAL CUAL (cell supernatant, culture fluid...)From each well of cell culture plate, on the third post-infection day (PID-3) of passage-1 (P-1), 50 μl of supernatant was taken and tested for SARS-CoV-2 using real-time RT-PCR for E and RNA-dependent RNA polymerase (RdRp) (2) genes
Similar testing was repeated on the cell supernatant of passage-2(P-2) at PID-4 for observing viral copy number.
Next-generation sequencing was performed on SARS-CoV-2 positive clinical samples (100 μl) included in the study and the tissue culture fluid (50 μl) of virus isolates at PID-3
NADA DE AISLAMIENTO. NADA. LO MISMO, SECUENCIARON EL CULTIVO TAL CUAL. Aparece de nuevo nuestro "amigo" el QIAamp viral RNA mini kit.Detection of SARS-CoV-2 in suspected samples:
The viral RNA was extracted from the TS sample using the Magmax RNA extraction kit (Applied Biosystems, USA) as per the manufacturer's instructions.
The extracted RNA was immediately used for testing the presence of SARS-CoV-2 using the real-time RT-PCR protocol published by the WHO
NGS of SARS-CoV-2 from India - Phylogenetic analysis and molecular characterization:
The total RNA of three positive TS specimens from Kerala, was extracted from 250-300 μl of the SARS-CoV-2 real-time RT-PCR positive samples.
QIAamp Viral RNA extraction kit (QIAGEN, Hilden, Germany) was used according to the manufacturer's instructions.
The extracted RNA was further quantified using a Qubit RNA High-Sensitivity kit (Invitrogen, USA).
RNA libraries were prepared as per the earlier-defined protocol and quantified using
La conclusión principal es que el virus no ha sido aislado, incumpliendo no sólo esta parte de los postulados de Koch (el aislamiento) sino incumpliendo las recomendaciones acerca de la propia NGS.Sample preparation.
Theoretically, any type of sample can be analyzed using the metagenomic approach, including seawater , blood , horse feces , stool [20•, 68, 69, 70, 71], marine sediments , coral tissues [72, 73], and hot springs . Because viral genomes are relatively short, bacterial or eukaryotic nucleic acids can severely interfere with the isolation and detection of viral DNA or RNA that typically represents only a small fraction. Thus, removal of non-viral nucleic acid is necessary [64••, 75].
Todos los mass-mierda atacaron descaradamente a KyleEEUU - Protestas, Tiroteos:
En los Vídeos se muestra como KYLE RITTENHOUSE se defiende desde el suelo de una turba que quiere lincharlo disparando para salvar su vida.
Protestas en Kenosha: Kyle Rittenhouse, el joven de 17 años acusado de matar a 2 personas en las manifestaciones contra la brutalidad policial en Wisconsin.
La guerra civil en EEUU está cada vez más cerca, Pero tienen una cosa que Europa carece RESISTENCIA