Thermo Fisher Scientific VetMAX C. burnetii Absolute Quant Kit Operating instructions

Brand: Thermo Fisher Scientific

Model: VetMAX C. burnetii Absolute Quant Kit

Type: Operating instructions

For Veterinary Use Only. For In Vitro Use Only.

INSTRUCTIONS FOR USE

VetMAX™ C. burnetii Absolute Quant Kit

TaqMan® real-time PCR for detection and quantification of Coxiella burnetii, responsible for Q fever

Catalog Number FQPAQ

Doc. Part No. 100020367 Pub. No. MAN0008368 Rev. B.0

Technology

Specie(s)

Nucleic acid isolated from matrices

Test type

Real-time PCR (DNA)

–Duplex

–Endogenous IPC

Bovines

Small ruminants

(sheep, goat)

Swabs (cervical, vaginal and placental)

Milk

Vaginal mucus

Amniotic fluid

Organs (placenta or fetal tissue)

Individual

Pool of 3 samples

(depending on matrices)

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,

clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.

WARNING! POTENTIAL BIOHAZARD. Read the biological hazard safety information at this product’s page at

thermofisher.com. Wear appropriate protective eyewear, clothing, and gloves.

Information about the product

Description of the product

Q fever (Query fever) is a zoonosis caused by Coxiella burnetii, an obligate intracellular bacteria that can infect many animal species as

well as humans. In ruminants, this disease is mainly associated with the appearance of reproductive problems. Coxiella burnetii causes

prematurity, low birth weights and miscarriages.

The Applied Biosystems™ VetMAX™ C. burnetii Absolute Quant Kit is a molecular diagnostic tool for Q fever. It enables specific

detection of the Coxiella burnetii bacteria by real-time PCR. It can be used in a qualitative application (presence or absence of bacteria) or

an absolute quantitative application (concentration of bacteria per sample unit).

Each DNA sample obtained after extraction is analyzed in a single well: the same well is used for specific detection of the bacterial

DNA of Coxiella burnetii and for the detection of an IPC (Internal Positive Control). A positive IPC reflects both the efficiency of

extraction and the absence of inhibitor in the samples.

It can be used with bacterial DNA extracted from swabs (cervical, vaginal and placental), milk, vaginal mucus, amniotic fluid or

organs (placental or fetal tissue). For swabs only, the analysis can be performed on pools of 3 samples.

Complete protocols for DNA extractions from these matrices are available upon request from Technical Support.

Kit contents and storage

The VetMAX™ C. burnetii Absolute Quant Kit comes in a case containing the different components. Upon receipt, the entire kit

should be stored at −30°C to −10°C. After thawing for the initial use, follow the recommendations for storage of each component in the

following table:

Component Description Volume

(100 tests)

Storage

Upon receipt

After initial

use

3 - Mix Cox b.

(Green tube)

Mix for TaqMan® PCR. Contains:

•The detection system for

Coxiella burnetii

target: forward and

reverse primers, as well as a TaqMan® probe labeled

FAM™ - TAMRA™.

•The detection system for IPC: forward and reverse primers,

as well as a TaqMan® probe labeled VIC™ - NFQ

(NFQ = Non-Fluorescent Quencher).

•The buffer and the PCR enzyme, as well as Uracil

N-Glycosylase.

2 × 1000 µL −30°C to −10°C +2°C to +8°C

4b - Standard PC. Cox b.

(Brown tube)(1)

The kit’s standard for

Coxiella burnetii

quantification. This is

plasmid DNA for dilution to prepare a quantification range. Its

use enables absolute quantification of an unknown sample.

100 µL −30°C to −10°C −30°C to −10°C

(1) The tube of 4b - Standard PC. Cox b. should be used with caution; the high DNA target concentration can be a source of contamination.

Extraction and amplification controls

The VetMAX™ C. burnetii Absolute Quant Kit contains 1 control, enabling validation of the amplification of the bacterial DNA and

quantification of the samples:

2 VetMAX™ C. burnetii Absolute Quant Kit Instructions for Use

4b - Standard PC. Cox b.: Coxiella burnetii positive control

Positive control, already extracted, to be amplified during real-time PCR. A positive result within the specified Ct range is used to

validate the amplification of the Coxiella burnetii target by real-time PCR.

This standard is quantified at 3.00 × 107 copies GE/mL. Dilution of the range standard, followed by amplification of this range during

real-time PCR, enables quantification of the samples.

Validation of nucleic acid extraction for each sample is done by detection of an endogenous IPC (Internal Positive Control), present

in every sample. A positive IPC result with a compliant value in a sample validates the extraction of this sample, whether positive or

negative for the target pathogen, thus eliminating false negatives and verifying the inhibitor effect.

We recommend that two negative controls be performed to confirm correct analysis:

NCS: negative extraction control

This control consists of reagents used in the extraction without addition of the sample (it can be replaced by the buffer used in the

sample preparation or by DNase/RNase-free water), which undergoes the same treatment as the samples: nucleic acid extraction and

then real-time PCR.

A negative result for Coxiella burnetii and for the IPC confirms the absence of contamination during the extraction and the real-time PCR.

NC: negative amplification control

This control consists of 20 µL of the real-time PCR mix and 5 µL of DNase/RNase-free water undergoing real-time PCR.

A negative result for all targets (Coxiella burnetii and IPC) confirms the absence of contamination during PCR reaction preparation.

Materials required but not provided

Unless otherwise indicated, all materials are available through thermofisher.com.

• Precision micropipettes (range of 1 µL to 1000 µL) with DNase/RNase-free filtered tips

• DNase/RNase-free water

• 1X TE buffer

• 1X PBS buffer

• A real-time PCR thermal cycler capable of detecting the following fluorophores:

– FAM™ (maximum emission: λ515 nm)

– VIC™ (maximum emission: λ554 nm)

• Optical-quality consumables compatible with the thermal cycler used:

– PCR plates with 96 wells, PCR strips (8 or 12 wells), microtubes or capillaries

– Films or caps suitable for closing

Analysis procedure

The real-time PCR reaction volume is 25 µL:

• 3 - Mix Cox b.: 20 µL per analysis.

• Extracted DNA: 5 µL per analysis.

For a quantitative application: prepare the quantification range using 4b - Standard PC. Cox b.

DNA extraction

It is necessary to isolate the DNA from the samples for real-time PCR analysis. To facilitate the quantification of non-liquid samples, it

is recommended to work with a swab eluate or with supernatant from crushed organ made in 1 mL of PBS 1X.

NOTE: For information about extraction methods that are compatible with the VetMAX™ C. burnetii Absolute Quant Kit, please contact

Technical Support.

Preparation of the quantification range

Take appropriate precautionary measures to avoid contamination of the real-time PCR mix 3 - Mix Cox b when performing this step.

Serially dilute the DNA from 4b - Standard PC. Cox b. to 1:10, preferably in 1X TE buffer for better storage (or in DNase/RNase-free

water) to constitute the 5 range points:

Range point

Concentration

Preparation of range point

Point 1 (Standard PC. Cox b.10

−1

)

3.00 × 10

copies GE

(2)

/mL

20 µL of 4b - Standard PC. Cox b. + 180 µL of 1X TE

Point 2 (Standard PC. Cox b.10

−2

) = EPC Cox b.

(1)

3.00 × 10

copies GE/mL

20 µL of Standard PC. Cox b. 10

−1

+ 180 µL of 1X TE

Point 3 (Standard PC. Cox b.10

−3

)

3.00 × 10

copies GE/mL

20 µL of Standard PC. Cox b. 10

−2

+ 180 µL of 1X TE

Point 4 (Standard PC. Cox b.10

−4

)

3.00 × 10

copies GE/mL

20 µL of Standard PC. Cox b. 10

−3

+ 180 µL of 1X TE

Point 5 (Standard PC. Cox b.10−5) 3.00 × 102 copies GE/mL 20 µL of Standard PC. Cox b. 10−4 + 180 µL of 1X TE

Serial dilution factor: 1:10

(1) For qualitative application, it is not necessary to add the entire range of quantification. A single dilution point for this range is used as the amplification control:

EPC Cox b.

(= DNA standard Cox b. 3.00 × 105).

(2) Genome Equivalent.

Aliquot the DNA standards below 30 µL and store them below −16°C for the entire period of kit storage. More than 3 cycles of

freezing/thawing standard DNA is not recommended.

VetMAX™ C. burnetii Absolute Quant Kit Instructions for Use 3

Preparation of the real-time PCR

1. Create an analysis plan for distribution of the mixes and samples. Keep the quantification range or the EPC away from the other

samples, if possible.

2. Thoroughly mix the 3 - Mix Cox b. tube by gentle agitation, then centrifuge briefly.

3. Add 20 µL of 3 - Mix Cox b. to each well of the PCR plate, PCR strip or capillary used.

4. Add the DNA samples and controls to each reaction mix according to the predefined analysis plan:

Type of analysis

Component

Sample volume

Sample for analysis

DNA extracted from the sample

5 µL

Quantification range

Dilution range of 4b - Standard PC. Cox b.

5 µL

Positive amplification control

(1)

EPC Cox b. (= standard PC. Cox b. 10

−2

)

5 µL

Negative extraction control (NCS)

Extracted NCS

5 µL

Negative amplification control (NC)

DNase/RNase-free water

5 µL

(1) This point replaces the range of quantification for qualitative applications.

5. Cover the PCR plate, PCR strips or capillaries with an adhesive film or suitable caps.

Amplification by real-time PCR

1. Create the following detectors on the thermal cycler:

Reporter

Quencher

COXB (Coxiella burnetii)

FAM

™

TAMRA

™(1)

IPC COXB

VIC

™

NFQ (Non-Fluorescent Quencher)

Passive reference: ROX

™(1)

(1) The fluorophores TAMRA™ and ROX™ must be entered for real-time PCR analysis if the thermal cycler is capable of detecting them. For other thermal

cyclers, absence of detection of these fluorophores does not compromise the analysis by real-time PCR.

2. Assign the COXB detector and the IPC COXB detector to each sample well used in the analysis.

3. Assign the quantification values (copies/mL) for each quantification range point.

4. Set up the following real-time PCR program for the analysis:

Step repetitions

Temperature

Duration

Step 1

1×

50°C

2 minutes (02’00”)

Step 2

1×

95°C

10 minutes (10’00”)

Step 3 45×

95°C

15 seconds (00’15”)

60°C

(1)

1 minute (01’00")

(1) Collection of fluorescence data during the 60°C – 1 minute phase

5. Place the PCR plate, the PCR strips or the capillaries in the thermal cycler and run the real-time PCR.

Analysis of the results

Analysis of the raw data

Refer to the recommendations of the thermal cycler manufacturer for analysis of the raw data.

1. Set the threshold limits separately for each real-time PCR target.

2. Interpret the results based on the Ct values of the samples for each detector according to the following recommendations.

Test validation

Validation of qualitative tests

The qualitative test is validated if the following acceptance criteria are met:

COXB detector

IPC COXB detector

Validation

EPC Cox b.

= C

COXB of Standard PC. Cox b.10

−2

±3C

t(1)

< 45 or C

> 45

(2)

PCR validated

NCS

> 45

Extraction step validated (no contamination)

Ct > 45 Ct > 45 PCR components validated (no contamination)

(1) Refer to the values listed in section 2.1 “EPC” of the Certificate of Analysis for the lot used for the test.

(2) The IPC value in the EPC should not be used to validate the test.

Additional validation criteria for quantitative tests

If not already performed, assign quantification values (copies/mL) to each quantification range point in the thermal cycler’s real-time PCR

software. The quantitative test is validated using the following acceptance criteria, as well as the qualitative test validation criteria.

COXB detector

PCR efficacy

Correlation coefficient R

Interpretation

Standard

quantification

range

At least 4 out of 5 of the range points validated

according to the conditions:

Ct = Ct QC COXB of Standard PC. Cox b. ±3Ct(1)

Between 85% and 115% R2 > 0.980 Quantification range

validated

(1) Refer to the values for the different points of the range listed in section 2.1 “EPC” of the Certificate of Analysis of the lot used for the test. An abnormal point

can be eliminated from within the range, but not from the limits.

thermofisher.com/support | thermofisher.com/askaquestion

thermofisher.com

31 May 2017

Interpretation of the results

Qualitative application

For each sample analyzed, the results should be interpreted as shown below:

COXB detector

IPC COXB detector

Interpretation

< 45

< 45 or C

> 45

Coxiella burnetii

detected

> 45

< 45

Coxiella burnetii

not detected

> 45

Not validated

Quantitative application

Calculating the concentration in the DNA sample

For the absolute quantification of a DNA sample in which the Coxiella burnetii target has been detected:

1. Make sure that the quantification range is validated.

2. Use the real-time PCR software to calculate the Coxiella burnetii target quantity for each sample using the quantification range values.

Calculating the concentration in the liquid sample before purification of nucleic acid

The calculation is performed from the quantification of the DNA sample obtained during PCR (in copies/mL), the volume of the test

sample for extraction (TS) expressed in µL and the elution volume (EV) of the extraction method expressed in µL:

Liquid sample quantification (copies C. burnetii / mL) = PCR quantification (copies/mL) × [EV (µL) / TS (µL)]

Calculating the quantification of organs from the quantification of crushed organ supernatant

Quantification per mg of organ based on the quantification of the crushed organ supernatant (copies/mL), the volume of buffer used

for crushing (CV) in mL and the quantity of sample (QS) in mg:

Organ quantification (copies C. burnetii / mg) = crushed organ supernatant quantification (copies/mL) × [CV (mL) / QS (mg)]

Procedure for handling non-validated samples

For the analyses described below, it is important to take into consideration the dilution factors applied over the course of the process

during sample quantification calculation.

1. Dilute the DNA of the non-validated sample as indicated: at a 1:10 dilution in 1X TE buffer.

2. Perform a new real-time PCR analysis with 5 µL of this dilution.

3. If the diluted DNA is positive for Coxiella burnetii or negative for Coxiella burnetii with a compliant IPC result, then the obtained

result is validated.

4. If the diluted DNA is negative for Coxiella burnetii with a non-compliant IPC result, the obtained result is not yet validated. In this

case, repeat the nucleic acid extraction using the sample pre-diluted to 1:10 in 1X PBS buffer before extraction.

5. If the result is still not validated, repeat the analysis on a new sample.

Documentation and support

Customer and technical support

Technical support: visit thermofisher.com/askaquestion

Visit thermofisher.com/support for the latest in services and

support, including:

• Worldwide contact telephone numbers

• Order and web support

• User guides, manuals, and protocols

• Certificates of Analysis

• Safety Data Sheets (SDSs; also known as MSDSs)

NOTE: For SDSs for reagents and chemicals from other

manufacturers, contact the manufacturer.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant

their products as set forth in the Life Technologies' General

Terms and Conditions of Sale found on Life Technologies'

website at www.thermofisher.com/us/en/home/global/

terms-and-conditions.html. If you have any questions, please

contact Life Technologies at thermofisher.com/support.

Laboratoire Service International (LSI) | 6 Allée des Écureuils |

Parc Tertiaire du Bois-Dieu | 69380 Lissieu, France

Translated from the French Pub. No. MAN0007807 Rev. B.0.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR

ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,

PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH

OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history of Pub. No. MAN0008368 (English)

Revision

Date

Description

B.0 31 May 2017

Changed IPC. Modified standard concentration

unit. Updated to the current document template,

with associated updates to the warranty,

trademarks, and logos.

A.0

30 January 2014

Baseline for revision history

Limited Use Label License No. 460: PCR Veterinary Diagnostics. Notice to

Purchaser: For veterinary diagnostic services including the reporting of such

services for a fee and for research purposes only. Human diagnostic uses

require a separate license from Roche.

property of Thermo Fisher Scientific and its subsidiaries unless otherwise

specified. TaqMan is a registered trademark of Roche, used under permission

and license.

Thermo Fisher Scientific VetMAX C. burnetii Absolute Quant Kit Operating instructions

Thermo Fisher Scientific VetMAX C. burnetii Absolute Quant Kit Operating instructions

Thermo Fisher Scientific VetMAX C. burnetii Absolute Quant Kit Operating instructions

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