Met behulp van een Pan-Viral Microarray Assay (Virochip) op klinische stalen scherm voor virale pathogenen

This procedure uses the Viro chip, a pan viral microarray assay to analyze clinical samples for both known and unknown viruses. First nucleic acids are extracted from clinical samples. Next, the extracted RNA is reversed transcribed using random primers.

Second strand CD NA synthesis is performed and the CD NA library is amplified with random PCR. Then the amplified cd NA is labeled with fluorescent dyes and hybridized to a viral chip microarray. The final step is to scan and analyze the microarray.

Ultimately, results can be obtained that show the presence or absence of a virus or several viruses through analysis of the chip microarray. Broad spectrum detection methods such as a viral chipp microarray are needed in diagnostic microbiology because common clinical syndromes such as pneumonia, are not specific to any individual pathogen, and because of the continual emerging threat of novel viral pathogens such as the SARS Coronavirus and 2009 novel H one N one influenza virus, This method can help answer key questions in the field of diagnostic microbiology, such as what percentage of undiagnosed infections in the clinical setting may be caused by as yet unidentified novel viruses. The implications of this technique extend toward better diagnosis of acute clinical illness in the hospital because we are testing for hundreds to thousands of potential pathogens simultaneously.

We first had the idea for this method in 2002 when we decided to apply the newly developed technology of microarrays pioneered in the laboratory of Dr.Joseph DeRisi at UCSF to the detection of viruses. Our first real world test case was using the viral chip to identify a novel coronavirus responsible for SARS in 2003, demonstrating the procedure from start to finish will be a technician from my laboratory who will demonstrate the use of the viral chip assay on a nasal swab sample from a child to diagnose the cause of her acute respiratory illness. Nasopharyngeal swab samples are collected in sterile containers containing viral transport, liquid media, and stored frozen in a minus 80 degrees Celsius freezer.

In a biosafety hood, allow the tube containing the sample to thaw and briefly stir the swab in the media before disposing of the swab in a biohazardous waist bin. Then aliquot 200 microliters of the sample into a 1.5 milliliter tube. Pass the sample through a 0.22 micron spin filter to purify for viral particles and into a fresh 1.5 milliliter tube.

Next, use the Turbo DNA's kit from Ambien per manufacturer's instructions to degrade host genomic DNA and purify for protected encapsulated viral nucleic acid. Follow this with RNA extraction, using the XMO ZR viral RNA extraction kit or Qiagen ultrasound's virus kit per the manufacturer's instructions. Measure the concentration of the extracted RNA using a NanoDrop nucleic acid spectrophotometer.

RNA concentration is preferably at least 10 nanograms per microliter, but will vary from sample to sample using a thin wall 200 microliter tube. Add one microliter of primer A at a concentration of 40 PICA moles per microliter to four microliters of extracted RNA. Heat the sample to 65 degrees Celsius for five minutes in a thermocycler, and then let the tube cool to room temperature for five minutes.

Next, add five microliters of a master mix containing two microliters five times RT buffer, one microliter 12.5 millimolar, DNTP one microliter water 0.5 microliters, 0.1 molar DTT and 0.5 microliters superscript three, reverse transcriptase in a thermocycler. Incubate the tube at 42 degrees Celsius for 60 minutes. Then heat the sample to 94 degrees Celsius for two minutes.

To abort the reverse transcriptase reaction, call to 10 degrees Celsius for two minutes and pulse centrifuge for five seconds. Next, add five microliters of quease mix consisting of one microliter five times quease buffer, 3.8 microliters water and 0.15 microliters quease the second strand synthesis. Ramp the thermocycler up from 10 degrees Celsius to 37 degrees Celsius over eight minutes.

Hold at 37 degrees Celsius for eight minutes. Then heat to 94 degrees Celsius for two minutes to inactivate the quease and call to 10 degrees Celsius at this point. If desired, store the sample containing 15 microliters CDNA at minus 20 degrees Celsius.

First, add five microliters of CD NA sample to 45 microliters of a master mix consisting of five microliters 10 times PCR buffer, one microliter 12.5 millimolar, DNTP one microliter 100 pomo per microliter primer, B one microliter cleantech LA and 37 microliters of water. Then run the PCR protocol as follows, 94 degrees Celsius for two minutes, followed by 25 cycles of 94 degrees Celsius for 30 seconds. 50 degrees Celsius for 45 seconds and 72 degrees Celsius for one minute.

Then an an kneeling step of 72 degrees Celsius for five minutes, followed by a hold at 10 degrees Celsius after PCR. Check the PCR product and a 1.5%AOSE electrophoresis gel. A smear from approximately 200 to 1000 base pairs should be visualized.

To incorporate AAD DNTP, add five microliters of viral PCR product to 45 microliters of a master mix consisting of five microliters 10 times PCR buffer, one microliter 12.5 millimolar, A-A-D-N-T-P one microliter 100 p of multiple microliter primer, B one microliter ClinTech LA and 37 microliters water. Run the PCR protocol as follows, 94 degrees Celsius for two minutes, 15 cycles of 94 degrees Celsius for 30 seconds. 50 degrees Celsius for 45 seconds.

72 degrees Celsius for one minute and a kneeling step of 72 degrees Celsius for five minutes and hold at 10 degrees Celsius. Then clean the new PCR product with the DNA clean and concentrator three kit per the manufacturer's instructions and dute in 10 microliters of elution buffer. Add one microliter of one molar bicarbonate and one microliter si three to the 10 microliters of PCR product and incubate for one hour in the dark.

Next, again, clean the S labeled sample with DNA clean and concentrator five kit and elute in 12 microliters of elution buffer. Use 1.5 microliters of sample to check CDNA concentration and the amount of dye incorporated on a NanoDrop spectrophotometer in a PCR strip tube at one microliter 25 times fragmentation buffer five microliters five times blocking buffer 9.5 microliters or amount necessary for a normalized final concentration of 10 peak or moles per microliter of SI three labeled sample and water to a total volume of 25 microliters. Then add 25 microliters of two times GX hybridization buffer, and spin down briefly to remove bubbles.

Place a new gasket slide in the hybridization chamber. Load 40 microliters of the sample onto the gasket slide. Next place the Agilent printed viral array with the side marked Agilent down on top of the gasket slide and fasten the screw tightly.

Place the hybridization chamber in a 65 degrees Celsius oven and hybridize overnight using a slow rotation of 10 rotations per minute to wash the arrays. First preheat buffer two to 37 degrees Celsius while heating. Fill a 500 milliliter plastic container with approximately 250 milliliters of buffer.One.

Remove the array from the hybridization chamber, submerge the array and separate it from the gasket slide. Transfer the array to the clean container and wash in buffer one for one minute. Then using a separate 500 milliliter plastic container.

Wash the array in preheated buffer two for one minute. After washing, slowly remove the array from buffer. Two, being careful to avoid bubbles.

Use a kim wipe to wick away extra moisture while being careful to avoid directly touching the active side of the array. Finally, place the slide into the slide holder Agilent side up and insert into the array scanner. In this procedure, we use an Agilent two micron DNA microarray scanner.

Scan the SI three labeled array at five microns or two microns according to the instrument standard scanning protocol. Analyze viral chipp microarrays by cluster analysis or the automated RO chipp analysis program e unpredic. Using the Viro Chipp microarray 2009 novel influenza H one N one virus is readily detected in the nasal swab sample obtained from a child with influenza-like illness.

In addition, we have shown that the viral chip is capable of identifying not only respiratory viruses, but also a broad range of viruses from a variety of different tissues and bodily fluids. Some examples of virus detection in whole blood and serum using the vichi as shown here. These are ere calls corresponding to viro chip microray from blood samples, either whole blood or serum taken from four patients for each sample, the top two virus predictions are shown with the top virus prediction having the lowest P value.

For example, in the sample highlighted in light blue, the top prediction for that sample is dengue virus type one. While the second ranked prediction is dengue virus type two, the actual virus in that whole blood sample was indeed confirmed to be dengue virus type one in agreement with the dict results. Another application of the viro chip is in novel pathogen discovery.

Some examples include the discoveries of sars coronavirus X-M-R-V-A, OME retrovirus linked to prostate cancer and chronic fatigue syndrome in humans and HTCV, A novel human cardio virus associated with respiratory and diarrhea infections in children Once mastered. This technique can be done in 12 hours if it is performed properly. While attempting this procedure, it is important to remember to ensure the availability of all necessary equipment and reagents following this procedure.

Other methods like specific viral PCR amplification and deep sequencing can be performed in order to confirm and follow up unusual viral chip hybridization patterns or viral chip results that suggest the presence of a novel virus After its development. This technique paved the way for researchers in the field of oncology to explore the association of novel viruses with cancer in the fields of biodefense and clinical microbiology to develop condensed micro based assays for rapid pathogen diagnosis, and in the field of virology for characterization of novel viruses and their association with disease. After watching this video, you should have a good understanding of how to perform all steps of the viral chip microray assay, a broad spectrum surveillance test to detect viral pathogens in clinical samples, all the way from nucleic acid extraction, from clinical samples to microray hybridization and analysis.

Don't forget that working with clinical samples is considered biohazardous and nucleic acid extraction should be performed in laboratories rated at biosafety level two or higher.