Most of the projects we work on, whether profiling host gene expression or micobial communities, often yield at a candidate list of genes or organisms that we believe are linked to some phenotype. Increasingly, we are interested in methods and technology that let us quantitiatively measure these features, particularly in a way that is portable and could be deployed 'stall-side' in a barn just as easily as at a lab bench. For this, we've been using the Biomeme platform for end-to-end QPCR-based detection in the field
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Before starting

The protocols below detail how we’ve been using Biomeme Inc’s bulk reagents for QPCRs. Once you’ve optimized the assays using bulk reagents, assays can then be manufactured as room-temp stable QPCR assays for use in the field. For the most part, we keep our mastermix and PCR conditions the same across all assays. The variations come with the primer/probe design and optimizing extaction of DNA or RNA from different sample types. So far, we’ve had good success with the primers/probes listed below and with extraction of nucleic acid from nasal and skin swabs.

Devices

The hand-held QPCR device made by Biomeme, called the ‘Two3’, can accomodate three PCR tubes and can detect two colors in each tube: FAM and Cy5 (or ATTO647N, which is brighter than Cy5, but more costly to order). Biomeme will soon be releasing the ‘three9’ which, as the name suggests, accomodates 9 tubes and three colors/tube: FAM, Cy5/ATTO647N and TexasRed. ATTO647N is the brightest channel on the three9, so consider good designing the target you anticipate being present in the lowest concentration and/or least sensitive PCR to that channel. FAM is the next brightest, followed by TexasRedX. If you are designing a triplex assay for use with the three9 that includes a positive control, use TexasRedX for the positive control.

Preparing mastermix

Resuspend bulk lyophilized LyoDNA, LyoRNA or LyoGreen master mix with 135 ul of PCR grade water + 18 ul of glycerol. This is a 10x stock. Addition of glyceral allows leftover mastermix to be frozen for reuse later. LyoRNA master mix contains a reverse transcriptase for QPCR assays where the target is a RNA molecule. Each resuspended vial contains enough mastermix for 60 reactions of 20ul/rxn.

Reaction set-up with LyoDNA or LyoRNA

A typical QPCR reaction on the biomeme platform contains the following components

Reagent vol (ul)
20x primer/probe 1
10x LyoDNA, LyoRNA or LyoGreen mastermix 2
isolated DNA or RNA 5-17
water qs to 20

Run set-up

  • To begin, launch the Biomeme app on the iPhone connected to your device.
  • To run any assays, you’ll need to set up a data folder by selecting the ‘data management’ button on the main screen. Name your folder, create any subfolders if necessary and save.
  • Back on the main screen, choose ‘protocol management’ to set up a protocol that will contain your thermocycling conditions and a standard curve (optional). We’ve always used the standard thermocycling conditions that come pre-set on the device as follows:
Temp (C) Time (min:sec)
45 10:00
95 10:00
95 0:15
60 1:00

Bold steps are repeated for 40 cycles total

  • Give your protocol a name and select ‘save’ in the upper right corner of the app
  • If you want to set-up a standard curve, select your protocol, scroll to the bottom, and select standard curve.
  • Currently, standards can only have three points. Enter the values for your 3 point standard curve. Since the Two3 device sees a red and green channel, you have to enter 3 standard values for each channel. Enter zero or one for each point in the channel you’re not using.

  • To begin a run, go back to the main screen of the app and choose ‘start run’. Choose your data folder, then your protocol, then select the run type (standard curve, qualitative, etc). Hit run and follow the on screen instructions to begin the run

Avian assays

Tracheal swabs

All avian assays begin with tracheal swabs collected in brain-heart infusion (BHI) media. We typically have 11 swabs all stored in a single vial containing 5.5 ml of BHI media. These swabs could be from multiple birds or from a single bird sampled multiple times, and this vial can be assayed fresh or after storage at -80C. Here’s our procotol for rapid, 1 minute DNA extraction using Biomeme reagents:

  1. thaw vial containing BHI media and swabs
  2. take 100 ul aliquot of sample for DNA or RNA extraction (might be quite viscous due to mucous)
  3. add 100 ul sample to 1 ml of Biomeme Lysis Buffer (BLB) (if you plan to assay for IBV, use BLB from RNA kit)
  4. with a 1cc syringe affixed to a Biomeme extraction column, push the extraction sample mix up and down 10 times
  5. after expelling the volume the 10th time, wash the column by drawing up 500 ul of Biomeme Protein Wash (BPW) 1 time, then expel again.
  6. draw up 1 ml of Biomeme Wash Buffer (BWB) and expel volume
  7. draw up 1 ml of Biomeme Drying Wash (BDW) and expel volume
  8. remove and discard the 1cc syringe, and attach a new 50cc syringe to the same column. Pump up and down with air vigorously to dry the column.
  9. remove and discard the 50cc syringe. Connect new 1 cc syringe to the column and draw up 125 ul of Biomeme Elution Buffer (BEB).
  10. Expel the elution buffer into a final collection tube. Typical elution volume is 50-70 ul. We use 5ul of this DNA for any of the assays listed below, which means a single eluate is enough for at least 9-10 QPCR assays.

Primers and probes

Infectious Bronchitis Virus (IBV)

name Sequence (5’ -> 3’)
fwd GCT TTT GAG CCT AGC GTT
rev GCC ATG TTG TCA CTG TCT ATT G
probe FAM-CAC CAC CAG AAC CTG TCA CCT C-BHQ-1

Mycoplasma gallisepticum and Mycoplasma synoviae (Mg/Ms)

The primer and probe sequences below are from Raviv and Kleven, 2009

name Sequence (5’ -> 3’)
Mg fwd TTG GGT TTA GGG ATT GGGA TT
Mg rev CCA AGGG ATT CAA CCA TCT T
Mg probe 5TexRd-XN-TGA TGA TCC AAG AAC GTG AAG AAC ACC BHQ-2
Ms fwd CTA AAT ACA ATA GCC CAA GGC AA
Ms rev CCT CCT TTC TTA CGG AGT ACA
Ms probe FAM-AGC GAT ACA CAA CCG CTT TTA GAA T-BHQ-1

Cutaneous Leishmaniasis assay

Lesion swab

For each patient, two sterile swabs (provided by Biomeme) are held together and used to swab the inner border of lesion (encircled 20x). This will be used for DNA targets (bacteria). Repeat with two new swabs (20x), and use this second set of swabs for RNA targets (host genes).

  1. Immerse swabs intended for DNA in 1ml of a 1:1 mix of BLB from DNA kit (0.5ml) and water (BEB; 0.5ml), twirl in media for 1 min, discard swabs.
  2. Immerse swabs intended for RNA in 1ml of BLB from the RNA kit, twirl in media for 1min, discard swabs
  3. Attach standard lab 1cc syringe to Biomeme filter tip. Pump lysate up/down 10x, expel volume
  4. Immediately move to BPW wash (1ml), pump up/down 1x, expel volume
  5. Immediately move to BWB wash (1ml), pump up/down 1x, expel volume
  6. Immediately move to BDW wash (1ml), pump up/down 1x, expel volume
  7. Pump air up/down repeatedly 10x, discard syringe but keep filter tip
  8. attach 25cc or 50cc syringe to the same filter tip. Pump up and down with air vigorously to dry the column.
  9. remove and discard the syringe. Connect new Biomeme ‘slip-tip’ 1 cc syringe to the column and draw up 150 ul of Biomeme Elution Buffer (BEB). Pump up/down 3x with BEB.
  10. Expel the elution buffer into a final collection tube. Typical elution volume is ~120ul. We use 17ul of this DNA for any of the assays listed below, which means a single eluate is enough for at least 6 QPCR assays.

Primers and probes

Skin-resident microbes

The primers and probes below are used for detection of Staphylococcus aureus (Saur), Streptococcus pyogenes (Spyo) and Leishmania braziliensis (Lbra)

  • Leishmania braziliensis primer/probes are from Weirather et al., 2011
  • Streptococcus pyogenes primer/probes were adapted from CDC here
  • Staphylococcus aureus primers and probes were provided by Biomeme Inc.
name Sequence (5’ -> 3’)
Saur fwd provided by Biomeme
Saur rev provided by Biomeme
Saur probe provided by Biomeme
Spyo fwd GCA CTC GCT ACT ATT TCT TAC CTC AA
Spyo rev ATT ACT GGT TTC CAA GAC ATT GTG AC
Spyo probe /56-FAM/CCG CAA CTC /ZEN/ ATC AAG GAT TTC TGT TAC CA/3IABkFQ/
Lbra fwd TGC TAT AAA ATC GTA CCA CCC GAC A
Lbra rev AAA TGG CAT ACA GAA ACC CCG TTC
Lbra probe /56-FAM/GCC TCT GGG /ZEN/ TAG GGG CGT TCT GCA A/3IABkFQ/

Human transcripts

The primers and probes below are for the detection of host transcripts present in cellular material on recovered from the swab

name Sequence (5’ -> 3’)
hIL1B fwd CAA AGG CGG CCA GGA TAT AA
hIL1B rev CTA GGG ATT GAG TCC ACA TTC AG
hIL1B probe /56-FAM/AGA GCT GTA /ZEN/ CCC AGA GAG TCC TGT/3IABkFQ/
hGZMB fwd GTA CCA TTG AGT TGT GCG TG
hGZMB rev CAT GCC ATT GTT TCG TCC ATA G
hGZMB probe /56-FAM/CCT CCA GAG /ZEN/ TCC CCC TTA AAG GAA GT/3IABkFQ/
h18S (+ control) fwd provided by Biomeme
h18S (+ control) rev provided by Biomeme
h18S (+ control) probe provided by Biomeme
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