How PaRTI-Seq® works
Learn how PaRTI-Seq® helps to save more than 50% of sequencing cost
Smaller sequencing solutions like MiSeq make it possible to test just sample of 1 patient but sequencing cost in that case is still extremely high and make it least favorable option for most patients today.Cost of test using sequencing depends on many factors like capital cost of sequencer, consumables and reagents, quantity of samples in a sequencing run, and etc., so total sequencing cost of pathogen detection can vary ten and even more times!
On the other hand for standard test output requires as many as 20M reads.PaRTI-Seq® utilizes Devin® filter which allows to deplete up to 95% of host DNA in 5 minutes and thus significantly increases the percentage of microbial sequencing reads so with PaRTI-Seq® output requires only 5 millions, sequencing reads per sample for the same sensitivity!
Extraction of DNA using Devin can reduce total cost of pathogen detection by sequencing by more than a half!
Depending on your needs, you can utilize the whole PaRTI-Seq® technology workflow or integrate just part of it.
For optimal sample preparation we recommend to use Devin® filter which allows to deplete host DNA interference more than 95% in just 5 minutes so you can save up to 75% of sequencing cost at the downstream sequencing.
Devin® filter is intended for use with a wide range of body fluids, including whole blood, plasma, swabs etc. We recommend to use Devin® filter immediately after blood or other liquid is drawn from the patient as long-term storage can greatly affect testing results.
For highly efficient DNA extraction, we recommend to use Devin® Enrichment Kit. Each Devin® Enrichment Kit already includes 24 Devin® filters for 24 reactions and can be used in manual or automated protocol.
Devin® blood fractionation syringe filter exploits the antifouling Zwitterionic coating technology to specifically capture the white blood cells (>99%), while allowing other blood components to flow through the membrane. This device provides fast and cost-effective solution to reduce the presence of human DNA while elevates the proportion of microbial pathogens in human blood.
We recommend to use Devin® filter immediately after blood is drawn as long-term storage of sample can affect accuracy of results.
Maximum storage time for specimen is 6 hours at ambient temperature. If sample is stored at 2-6 °C, storage time can be extended to 12 hours. Please, DO NOT filter frozen blood samples as it can cause clogging.
To start using Devin® filter Draw 3-10 mL of whole blood sample into the syringe. Open the filter package and securely attach the filter to the syringe on the one side then assemble the needle on the other. Hold the assembled syringe and filter vertically. Press down on the syringe plunger and gently push the sample through the filter to a 15 mL /50 mL falcon tube. Store the filtered sample in the vacutainer and use as soon as possible
Sample prep with Devin® filter
1. Draw 3-10 mL of whole blood sample into the syringe. Open the filter package and securely attach the filter to the syringe on the one side then assemble the needle on the other. Hold the assembled syringe and filter vertically. Press down on the syringe plunger and gently push the sample through the filter to a 15 mL /50 mL falcon (centrifugation) tube.
2. Centrifuge whole blood sample at 400 x g for 15 min at room temperature. Collect the plasma (upper layer clear phase) and transfer to a new 15ml / 50ml centrifugation tube.
3. Centrifuge plasma at 16000 x g for 15 min at room temperature. sample pellet will precipitation at the bottom of the centrifugation tube
Collect the precipitation and mark “Sample pellet”
Before starting microbial DNA extraction
The Nucleic Acid Extraction Kit is suitable for isolating bacterial DNA from whole blood, plasma or other body liquids. Extracted nucleic acids can be analyzed by downstream application, such as real-time PCR and/or next-generation sequencing.
Workflow is shown for whole blood as an example, other fluids have similar workflow
Before start DNA extraction, please make sure that you wear a mask and disposable gloves when handling as well use sterile consumables to avoid nuclease contamination. Reagent solution contains guanidine salt, avoid using bleach of any contact, flush with flowing water. Avoid vigorous shaking, in order to avoid excessive formation of foam. Do not expose opened reagents or plates to air. The evaporation would lead to pH change, or influence the extraction effectiveness
All reagents are colorless and transparent. Colored reagents indicate contamination, so you will need to replace with a fresh plate before proceeding.
If the temperature is below 20℃, please first place the reagent plate in an oven (perheated 42-60℃) for 5 to 10 mins.
All the Kit’s components can be safely transported without any specific conditions however for best results we suggest you to follow the storage conditions shown to you right upon receival of the package!
MICROBIAL DNA ENRICHMENT
1. Remove aluminum foil from the well carefully to avoid splashing and pipette well #4 buffer homogeneously. Transfer well #4 buffer into a new eppendorf and label MB on the cap.
2. Transfer well #2 and well #3 buffer into a new eppendorf and label WB1 Mix on the cap
3. Transfer well #5 and well #6 buffer into a new eppendorf and label WB2 Mix on the cap.
1. Put the eppendorf MB on the magnetic rack for 30 seconds to absorb magnetic beads, and recover supernatant to eppendorf WB2 Mix.
2. Remove the eppendorf MB from the magnetic rack. Add 500 µl WB1 Mix and vortex 2 seconds, then spin down.
Add 20 µl Lysozyme + 20 µl Proteinase K + 200 µl Incubation Buffer in sample pellet and pellet are resuspended well. React at 60°C for 10 minutes and vortex for 10 seconds per 4 minutes, then spin down after the reaction.
Add 300 µl 95%~100% EtOH. Vortex for 10 seconds and spin down.
Put the eppendorf MB on the magnetic rack for 30 seconds to absorb magnetic beads, and recover supernatant to eppendorf WB1 Mix.
Remove the eppendorf MB from the magnetic rack and add the whole buffer of the eppendorf Sample.
Vortex eppendorf MB for 5 minutes and spin down. Put the eppendorf MB on the magnetic rack for 90 seconds to absorb magnetic beads and discard supernatant.
Add 750 µl WB1 Mix. Vortex for 30 seconds and spin down. Put the eppendorf MB on the magnetic rack for 60 seconds to absorb magnetic beads and discard supernatant.
Add 750 µl WB2 Mix. Vortex for 30 seconds and spin down. Put the eppendorf MB on the magnetic rack for 60 seconds to absorb magnetic beads and discard supernatant. Repeat step 8 one more time.
Open the eppendorf-MB, and heat at 45°C for 10 minutes to dry the magnetic beads.
Add 50 µl of Elution buffer into Eppendorf MB and let them react at 45°C for 5 minutes, gently pipetting 5 times per minute.
After reaction completes, spin down and put the Eppendorf MB on the magnetic rack for 60 seconds to absorb magnetic beads. Collect the supernatant EB to a new Lobind tube and mark as the bacterial DNA product.
Extracted bacterial DNA product should be stored at -20°C and can be used for further analysis by downstream application, such as real-time PCR and/or next-generation sequencing.
|Reagent Type||Transportation ConditionStorage||Condition upon receival|
|Micronbrane Plate||Room Temperature||Room Temperature|
|IB||Room Temperature||Room Temperature|
|Proteinase K||Room Temperature (no longer than 5 days)||4C (functional refrigerator)|
|Lysozyme||Room Temperature (no longer than 5 days)||-20C (functional refrigerator)|
|Devin® filter||Room Temperature||Room Temperature|
Devin filter and DNA enrichment kit reagents can be safely transported and kept at the room temperature (25°C) in a dry place except Proteinase K (can be safely transported at room temperature no longer than 5 days then best stored at 4C) and Lysozyme (can be safely transported at room temperature no longer than 5 days then best stored at -20C)
With what types of samples can be used with Devin® filter?
- Devin can be used for all type of body fluids including whole blood, plasma, swabs, CSF, BALF, ascites and other body fluids containing significant human DNA contamination
For what kinds of microorganisms Devin® / PaRTI-Seq® can used for
- Devin® / PaRTI-Seq® can be used for detection of various microorganisms like bacteria, fungi, vir viruses etc. You can learn more about clinical test results .
What kind of sequencers can be used with PaRTI-Seq®
- PaRTI-Seq® can be exploited on any sequencing platform including Illumina, Nanopore.
Does Devin® filter utilize pore-size technique to deplete human DNA?
- No, Devin® filter exploits patented antifouling Zwitterionic coating technology to specifically capture the white blood cells (>99%), while allowing other blood components to flow through the membrane.
Can I use PaRTI-Seq® for manual / automatic workflow?
- Yes, PaRTI-Seq® can be both used for manual and automatic protocol.
What lab conditions are required for PaRTI-Seq®
- PaRTI-Seq® can be implemented in any lab setting, however sterile environment is not necessary.
How much blood/body fluid can be processed per Devin® filter
- Volume of specimen 3-10 mL
How long may I keep samples pre-processed with Devin® filter?
- PaRTI-Seq® can be implemented in any lab setting, however sterile environment is not necessary.
Which class of medical devices Devin® filter belongs to?
- Devin® filter is RUO and can be used as LDT in clinical setting under I/II class of medical devices.
How PaRTI-Seq® works to identify pathogens in 24h?
- PaRTI-Seq® utilizes optimized workflow of sample preparation method for next generation sequencing (NGS) and proprietary analytical methods, which provides precise test results within 24 hours upon sample receival. You can learn more about PaRTI-Seq®
What should I need for PaRTI-Seq® in my lab or clinical setting?
- Micronbrane produces and provides consumables, reagents, bioinformatic analytical software so you only will require any sequencer in your lab or clinical setting for using PaRTI-Seq® for rapid pathogen detection within 24H.