sop 3.6 revised fast-track respiratory pathogens · pdf filesop 3.6 revised - fast-track...

Fast-Track Respiratory Protocol

Page: 1 of 12

Author: Sandra Dollet / Stephane Pouzol

Document type: PROTOCOL

Document Number: SOP 3.6

Release: 1.2 - revised Nov 2011

CONFIDENTIAL

SOP 3.6 Revised - Fast-Track Respiratory Pathogens Protocol

Introduction After nucleic acids extraction from nasal swabs or aspirates as well as pleural effusions, the FTD respiratory pathogens test, based on six multiplex RT-PCR reactions, enables detection and identification of virus and bacteria responsible for pneumonia. FTD respiratory pathogens + , pathogens can be detected: influenza A (Flu A), influenza B (Flu B), coronavirus 229E (Cor229E), coronavirus OC43 (CorOC43), coronavirus NL63 (CorNL63), parainfluenza virus 1 (PIV1), parainfluenza virus 2 (PIV2), parainfluenza virus 3 (PIV3), parainfluenza virus 4 (PIV4), rhinovirus (Rhino), respiratory syncytial virus A et B (RSV), human metapneumovirus A and B (hMPV), adenovirus (Adeno), bocavirus (Boca), enterovirus and parechovirus (EV/PV), Staphylococcus aureus (S aur), Streptococcus pneumoniae (S pneu), Haemophilus influenzae type b (HiB) and Mycoplasma pneumoniae (M pneu). Rem: Internal Control is a naked RNA (BMV). FTD respiratory pathogens 21 PLUS , pathogens can be detected: influenza A (Flu A), H1N1, influenza B (Flu B), coronavirus 229E (Cor229E), coronavirus OC43 (CorOC43), coronavirus NL63 (CorNL63), coronavirus HKU1 (CorHKU1), parainfluenza virus 1 (PIV1), parainfluenza virus 2 (PIV2), parainfluenza virus 3 (PIV3), parainfluenza virus 4 (PIV4), human metapneumovirus A and B (hMPV),rhinovirus (Rhino), respiratory syncytial virus A and B (RSV), adenovirus (Adeno), enterovirus (EV), parechovirus (PV),bocavirus (Boca), Mycoplasma pneumoniae (M pneu), Chlamydia pneumoniae (C pneu), Staphylococcus aureus (S aur), Streptococcus pneumoniae (S pneu), Haemophilus influenzae type B (HiB). Rem: Internal Control is a virus (EAV).

Materials

- FTD respiratory pathogens+, 96 tests (Fast Track Diagnostics, ref. FTD-96-/12), 200 tests (Fast Track Diagnostics, ref. FTD-2-204+)

- FTD respiratory pathogens 21 PLUS,96 tests(Fast Track Diagnostics,ref. FTD-96/12)

- CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, ref. 185-5096) : others thermocyclers can be used

- Kit AgPath-ID™ One-Step RT-PCR Reagents (Applied Biosystems, ref. 4387391) - 96-well plates for CFX96, white shell/clear well, Hard-Shell PCR plates (Bio-Rad

Laboratories, ref. HSP9601)


- Microseal® ‘B’ Adhesive Seals (Bio-Rad Laboratories, ref. MSB1001) - Centrifuge (4 000 rpm)

RT-PCR procedure

• General information Each step of the experiment takes place in a dedicated room and the investigator must wear clean gloves, weekly lab coat, gown and daily sleeves. The process should be done in accordance with Good Laboratory Practice.

1. Keep all the reagents frozen at -20°C prior to u se.

2. As the 2X RT-PCR buffer from AgPathID Kit comes in a 14 ml bottle, it’s recommended during the first use, to make as much as possible 1.5ml aliquots to avoid freeze/thaw cycles.

3. 6 sterile 1.5ml tubes will be used for the 6 mixes preparation and must be therefore identified as “set 1”, “set 2” etc...

4. The primers and probes (PP) sets from Fast Track kit are specific for their own targets. Example: set 1 of FTD respiratory pathogens 21 plus contains primers and probes specific for Flu A, H1N1, Flu B and Rhinovirus.

5. Prepare a layout (based on the number of samples to test) for every experiment on a excel sheet as described in the example below:

• Mix preparation This step takes place in the MIX PCR room and mix preparation is carried out within a nucleic acids free hood. The investigator must wear a weekly lab coat, clean gloves and daily sleeves.


Rem: prepare 1 more sample/PP mix in order to have enough reagents to carry out the reaction for all the samples. .

1. Let reagents (water, 2X RT-PCR buffer and PP sets) thawing in ice prior to use except enzyme (keep it -20°C until to use).

2. Mix reagents according to the protocol describes above. According to the number of samples to process, you MUST prepare as many PP mix as your samples request. For example, for 16 samples to process: PP mix 1 = 17 x 1.5µl PP mix + 17 x 12.5µl 2X RT-PCR Buffer + 17 x 1µl enzyme. PP mix 2 = 17 x 1.5µl PP mix + 17 x 12.5µl 2X RT-PCR Buffer + 17 x 1µl enzyme. Etc,etc… PP mix 6 = 17 x 1.5µl PP mix + 17 x 12.5µl 2X RT-PCR Buffer + 17 x 1µl enzyme. Caution: You MUST change tips after each pipetting step.

3. Mix by pipetting up and down and briefly centrifuge the mix preparations.

• Preparation of the 96-well plate

This step takes place in the RNA ROOM and 96-well plate preparation is carried out within a dedicated RNA hood. The investigator must wear a weekly lab coat, clean gloves and daily sleeves.

1. Nucleic acids extracted from samples and controls are thawed prior to use.

2. Keep the 6 different mixes and nucleic acids refrigerate at +4°C (in ice).

3. For CFX96, use a 96-well plates, white shell/clear well. Caution: make sure of the plate orientation.

4. Homogenize nucleic acids by vortexing and briefly centrifuge prior to use.

5. Plate layout: example below

6. Dispense 15µl of each mix per well according to the layout.

7. Add 10µl of sample/control to the corresponding wells (raw by raw) and homogenize the mixture by pipetting up and down. Warning: there are 2 different positive controls in this experiment; positive virus control from line 1 to line 5 and bacteria po sitive control in line 6.

8. Seal the plate with an adhesive film before leaving the hood.

9. Spin plate at 4000 rpm for few seconds; ensure that there are no bubbles in wells.

10. Load the 96-well plate in the CFX96 instrument and watch for the right direction.


11. Launch CFX Manager software and select the appropriated program :

50°C 15 min 95°C 10 min 95°C 8 sec x 40 cycles 60°C 34 sec The program runs approximately 1:20h.


Results and interpretation

Once the assay is completed, you got this window on the screen (or click on quantification tab ). Then, you are ready to analyze your results.

First, you have to update the layout by carefully selecting the fluorophores (depending on the PP set), identifying targets and samples and indicating sample type.

1. Layout Updating

Once the run is over, the following page appears. Select ‘View / Edit plate ’ tab to update the layout.


a. Fluorophores selection:

i. Select a primers-probes set by selecting all the wells in the same line. Example for FTD 21 plus set 1:

ii. Click on “Select Fluorophores ” tab and apply the fluorophores according to the PP set selected. Example for FTD 21 plus set 1:


Table below is a summary of the fluorophores you may selected:

Note: YY and VIC have the same wavelengths of excitation and emission.

b. Targets identification

i. Assign a name to the targets. In order to easily and correctly analyze your data, it’s important to associate a pathogen to a fluorophore. You just have to write the correct pathogen name in the tab and validate by pressing enter or selecting the corresponding fluorophore tab. Example for FTD 21 plus set 1:

ii. Apply this procedure for each of the 6 primers-probes sets.


c. Assign Sample type and Sample Name:

i. Select the wells corresponding to the same sample or control and assign the correct type: “unknown” as sample, “positive control” or “negative control”.

ii. Write the name of your sample in the “Sample Name” tab (here is

Sample 1) and confirm by pressing ENTER or checking “Load” tab.

There is no need to fill the name if it’s a control.


d. Wells exclusion:

Select unused wells.

Then click on “Clear Wells” tab


2. Prepare Wells group a. In order to analyze and visualize samples set by set, it is necessary to make

groups. Click on the icon ‘Well Groups’ . The window ‘Well Group Manager ’ appears, click ‘Add ’ for the first group and then select samples for this group. Repeat for all sets.

b. Select OK to close the window ‘Edit / View plate ’.

c. According to the "Well Groups ” you’ve made, you are ready to analyze results set by set and fluorophores by fluorophores (by checking a single fluorophore).


3. Some examples with FTD respiratory pathogens + :

Bocavirus (set 4, ROX)

Positif

control

LOG SCALE : Bocavirus (set 4, ROX)

Late CT

Samples Negative

when baseline

higher than 102

Staphylococcus aureus (set 6, FAM)

L


• Tips for analysis

1. Baseline Set up:

2. Check internal control amplification/detection in samples and controls (it must be negative for positive control). Expected Ct range for IC is 26-31.

3. Check positive controls. The expected Ct should below 33Ct

4. Check negative controls. Only IC MUST be detected.

5. If you have a doubt to consider a sample as positive or not, double check the shape of the amplification curve in RFU (linear) and log scale.

S


6. Samples with CT above 35 (except Bocavirus whose baseline is 36 Ct) are considered as negative. In case of suspicious case, do not hesitate to retest the sample for this specific PP set.

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