表面上のヒトノロウイルスの検出のためのスワブサンプリング方法

The overall goal of our macrofoam based surface sampling method is to identify outbreak etiology, overall evaluation of environmental contamination dynamics of the virus, and effectiveness of remediation strategies for the norovirus. This protocol detailed how to collect swab sample from environmental surface and how these swabs should be stored and transported, and which laboratory protocol are recommended for the detection and typing of the norovirus from these swabs. Clear improvements of this environmental swab protocol for noroviruses is that we have determined the best type of swab material, how much norovirus can be recovered from hard surfaces, and that the protocol can be used effectively to sample larger surface areas.

In this video, two of my staff scientists, Geun Woo Park and and Preeti Chhabra will demonstrate the swab protocol for the detection and typing of noroviruses. To begin swabbing, proceed to the sampling area and move the swab across the sampling area with one stroke in the horizontal direction, one stroke in the vertical direction, and one stroke in the diagonal direction. Place each swab into a separate tube and tighten the cap securely.

Sample surfaces with stainless steel, sample a toilet seat surface, and sample a doorknob. Next, label one 15-milliliter tube and one RNA Midi column for each sample. Include a negative extraction control in each experiment.

Prepare an empty bottle to collect universal nucleic acid extraction or UNEX, buffer waste. To prepare the lysis solution for 10 swabs, mix 25 milliliters of UNEX buffer with 25 milliliters of PBST. Add 50 microliters of coliphage MS2 suspension to 50 milliliters of lysis solution.

Place a swab in a 15-milliliter tube, and add five milliliters of lysis solution. Mix the swab in the solution, and incubate the mixture at room temperature. Add five milliliters of 100%ethanol to each tube, and vortex the tubes for 10 seconds.

Carefully remove the swab from the 15-milliliter tube, pressing it gently against the side of the tube to remove excess liquid, and then discard the swab. Carefully transfer 4.5 milliliters of the UNEX ethanol mixture onto a Midi column, and centrifuge the column. After centrifugation, discard the filtrate.

Load another 4.5 milliliters from the same mixture onto the same column, then centrifuge the column. Discard the filtrate. For the first wash, add 3.5 milliliters of 70%ethanol onto the Midi column, and centrifuge the column.

After the spin, discard the filtrate. For the second wash, add another 3.5 milliliters of 70%ethanol onto the Midi column. Centrifuge and discard the filtrate.

For the dry spin, centrifuge the Midi columns to remove all traces of ethanol. Place the Midi column in a new 15-milliliter centrifuge tube. Add 250 microliters of elution buffer onto the Midi column, and wait for one minute before centrifuging to obtain the highest to viral RNA recovery.

Add 500 microliters of RNA binding buffer to 250 microliters of RNA, and vortex the mixture for 10 seconds. Add 750 microliters of 100%ethanol, and vortex for 10 seconds. Next, label a spin column for each sample.

Load the 750-microliter sample onto a spin column, and centrifuge the spin columns. Discard the flow-through. Load the remaining 750 microliters onto a spin column, and centrifuge the columns.

For the prewash, add 400 microliters of RNA prep buffer to each spin column, and centrifuge the column. Discard the flow-through. For the first wash, add 800 microliters of RNA wash buffer to each spin column, and centrifuge the columns.

Discard the flow-through. For the second wash, add 400 microliters of RNA wash buffer to each spin column, and centrifuge them. Discard the flow-through.

For the dry spin, centrifuge the spin column to remove all traces of wash buffer. Carefully transfer the spin column to a clean 1.5-milliliter microcentrifuge tube. Add 25 microliters of elution buffer onto the spin column, and incubate the column for one minute at room temperature to increase viral RNA recovery from the column.

Collect the RNA by centrifuging the spin column at 10, 000 times G for one minute. Proceed directly to real-time PCR. Before continuing with multiplex real-time RTPCR, clean working surfaces, pipettes, and centrifuges with an RNase decontamination solution.

Thaw real-time PCR reagents on ice. Thaw the sample RNA on ice in a separate area. Determine the number of reactions, and prepare at least 10%more reagent for the master mix to control for losses during pipetting.

Next, vortex the individual master mix components for five seconds. Carefully mix the enzyme by flicking the tube. Briefly centrifuge the master mix components for five seconds.

Then, prepare the master mix for the RTPCR detection of norovirus G1 and G2 according to the kit instructions, and add norovirus-specific oligonucleotide primers and probes in a 1.5-milliliter microcentrifuge tube. Next, mix the master mix by pipetting seven times up and down. Aliquot 22 microliters of the master mix into each well of the real-time PCR plate.

Vortex the sample RNA for five seconds, and collect the contents by five-second centrifugation. Add three microliters of sample RNA positive controls and 10-fold serial dilutions of G1 and G2 RNA transcripts to the real-time PCR plate. Add three microliters of nuclease-free water into the node template control, or NTC wells.

Seal the plate with optical adhesive film. Carefully centrifuge the plate at 1, 300 times G for one minute to remove any air bubbles or liquid drops that may be present in the wells. Then, set up the real-time PCR, and enter the thermocycling conditions.

Determine the standard curves for both the G1 and the G2 transcripts. Convert the Ct values into RNA copies using the respective standard curve. Take the ratio of the volume of total RNA eluent and the total amount of RNA used for the real-time assay to calculate the total number of RNA copies per sample.

To genotype the norovirus-positive samples using hemi-nested conventional RTPCR, first prepare a master mix for the first round RTPCR, and dispense 20-microliter aliquots in pre-labeld PCR tubes. Add five microliters of RNA to each tube. After the first round RTPCR is completed, prepare a master mix for the hemi-nested second PCR round by dispensing aliquots of 23 microliters into new tubes.

Finally, add two microliters of the first round PCR product to each tube. The macrofoam-based swab sampling method was field tested on samples collected from a cruise ship that had reported cases of suspected norovirus gastroenteritis. Four of the 17 norovirus-positive swab samples could be genotyped, and all samples had identical G2 sequences.

Viral load determination allowed assessment of surfaces which were heavily contaminated, such as toilet seats. The relationship of cycle threshold, or Ct values, was determined by real-time RTPCR in the ability to sequence these samples. In total, 127 out of 217 swab samples tested positive for G2 norovirus.

Swab samples with Ct values between 24 and 27 and between 28 and 31 were genotyped at rates of 100%and 72.2%respectively. In contrast, only 22%and 1.6%of swab samples with Ct values between 32 and 36 and 37 to 40 were sequenced successfully. As we have demonstrated, this protocol allows to determine the level of environmental contamination during norovirus outbreaks, detect the possible cause of an outbreak when clinical samples are not available, and also, to determine the effectiveness of cleaning practices.

For the most accurate result, it is very important to follow this protocol precisely to minimize the variation of the results among the different samples.