Detection of Neurovirus in extracted RNA by one step real-time R-T-P-C-R with specific TECHMAN probes. But today we're gonna talk about neurovirus. This is in Lima, Peru and it's a group of the university and also Johns Hopkins University.
Norovirus, it turns out, is probably one of the mostec neglected causes of diarrhea. Our group has found that norovirus is second only coronavirus in terms of children's hospitalizations for diarrhea and also in terms of population based number of incidences of diarrhea. So why has NEUROVIRUS not been diagnosed before?
Well, the reason is that it's an RNA virus and we can't culture, so it takes special molecular biological methods to diagnose. These methods are that we use are R-T-P-C-R, and this is going to demonstrate that how to use and diagnose how to use the R-T-P-C-R to diagnose neurovirus. In-house Ciag GUINE method boom etal 1989 we used an in-house G guine ATE method for extraction of viral RNA for stool samples.
This method is an alternative to the commercial ca amp viral RNA kit. Both methods are based on the nucleic acid binding properties of silica particles. Combined with the anti nuclease properties of G Guad Dium Bio NA preparation of silica suspension for RNA extraction.
To prepare the silica particles, take 30 grams of silicone dioxide, add 250 millimeter of demineralized water and suspend the silicon by shaking. After 24 hours of sedimentation, remove 215 milliliters of the supinate and portion by suction at 250 milliliters of demineralized water and again, suspend the silicon by shaking. After another 24 hours, remove the supinate by suction and adjust the pH of the silica suspension to pH 2.0 using hydrochloric acid qu suspension of silica in glass bottles and autoclave extraction with g guin and silica.
We'll now look at the extraction of viral RNA using the silica particles prepared in a previous step. Store at minus 70 degrees Celsius until use. To make a 10%fecal suspension take approximately 0.1 gram of stool and complete with one milliliter of PBS before the extraction.
Lower load samples vortex for five seconds and then centrifuge at 4, 000 RPM for 10 minutes. In each reaction, a neurovirus positive stool sample as positive control and DEPC treated water in a micro centrifuge tube. Mix the following, one milliliter of L six buffer, 20 microliter of the prepared silica particles and 200 microliters of fecal extract.
Vortex briefly and leave the samples at room temperature for 15 minutes. Centrifuge for 15 seconds. Then wash the pellet twice with buffer L two, then two washes with 70%ethanol and one wash with acid on.
Dry the pellet in a dry heating block at 56 degrees Celsius for five minutes. Hydrate RNA in the silica pellet by adding 50 microliters of RNA free distillated water. Add one microliter of RNA sin vortex and leave at 56 degrees Celsius for 15 minutes.
Centipede for three minutes at full speed and collect a supinate temp containing the RNA. Quantify the samples using non drop technique and store extracted RNA at minus 70 degrees Celsius. Until use commercial extraction of viral RNA.
We are now going to demonstrate the RNA extraction from stools using the commercial available kay amp viral RNA. The kit contains the following silica gel MinPin column buffer, A-V-L-A-W one a W2 A VE and leafly carrier RNA.Briefly. The procedure is as follows.
Mix a portion of the buffer A VL with carrier RNA and vortex Pipe at the solution into 1.5 milliliter tubes at 114 microliters of stool suspension and mixed by postwar texting. Incubate at room temperature for 15 minutes. Pulse centrifuge the samples.
Add ethanol to the samples and mix by postwar texting for 15 seconds. Then briefly centrifuge, apply the solution to the spin column in a two minimally collection tube. Pull fuge.
Then place the spin column in a new collection tube. Wash the column with bound RNA using buffer A W one without carrier RAN centrifuge. Place column in a clean two milliliter collection tube and watch the column with buffer.
A W2 centrifuge at full speed place pin column in a clean 1.5 milliliter micro centrifuge tube and full speed centrifugation. And elevate the RNA using buffer a VE store viral RNA at minus 70 degrees Celsius until use detection of neurovirus in extracted RNA by one step real time PCR with specific techman probes to make a mouse mix for geno group one and Dina group two, keep your enzyme RT mix on ice during assay. Set up to kit contains the following PCO water mastery, two eggs, primers and probes for Dina.
Group one primers and probe for Dina Group two. And finally, the enzyme RT mix vortex H reagent before use QU to PCR water the master mix two X primers coke G one forward and reverse verse and probe ring one A and one B for UniGroup. One muscle mix and primes COG G two for VA verse and probe.
Ring two for Theo Group two master mix. And finally, add a enzyme artix to both the INO group muscle mixes. Vortex and alco 10 microliters of the appropriate muscle mix to the reaction wells.
In another cabinet, add five microliters of undiluted, RNA five microliters, G one and G two positive control RNA and five microliters negative control to the corresponding reaction wheels. Centrifuge deplete for 10 seconds at 6, 000 g. In the step one software shows advanced setup.
And in the experiment property screen, the experiment is identified as norovirus. The experiment type is presence or absence on the target screen. Enter the name of the target you want to detect in the PCR reaction plate.
Gina, group one and two reporter fam and quencher Tamara on the sample and replicate screen. Enter the number of samples and positive and negative controls included on the reaction plate and then select which samples or target direction to set up In run method. First, make a reverse transcription at 50 degrees Celsius for synthesis of complementary DNA.
Then raise the temperature to nine to five degrees Celsius to activate a hot start polymerase. Start the cycling by duration at nine to five degrees Celsius, followed by a combined an kneeling and amplification step at 60 degrees Celsius. Repeat a cycling four to five times return to the plate setup.
Click save a template and save the experiment. Click start run. The amplification plot screen allows you to view sample amplification as your instrument collects frozen data during a run as well as the plot contrast.
Normalized dive fluorescence and cycle number. In the initial cycles of PCR, there is little change in fluorescent signal. This defines the baseline.
An increase in fluorescence about the baseline indicates the detection of accumulated target. A fixed fluorescent threshold should be set about the baseline. The parameter CT threshold cycle is defined as the fractional cycle number at which the fluorescence passes the fixed threshold.
The amplification plot screen is useful for identifying, examining abnormal amplification such as increased fluorescence in negative control wells and absence of detectable fluorescence at an expected cycle. The kit method is reliable, yet requires an enormous amount of time and resource. Here we have shown an equally reliable method that is cost effective and uses readily available in country reagents, bypassing reliance on materials purchased abroad, all of which will ultimately lead to a more accessible diagnosis and rapid treatment of the most common viral causes of gastroenteritis in children Using the economic in-house method for isolating nucleic acids, which obtained similar results to those.
Obtained with the commercial kit from Kerogen, along with the TAC MAN, R-T-P-C-R, which was developed in our laboratory. We can detect a broad range of neurovirus infection. The assay has proven useful for routine diagnosis as it eliminates post amplification product processing, thus shortening the time of the assay.
Besides diagnosis, the protocol can also be used for clarifying the epidemiology of neurovirus infection, thus being useful for public health control of this disease.
