Thermo Fisher Scientific MycoSEQ Plus Mycoplasma User guide

For Research Use Only. Not for use in diagnostic procedures.

MycoSEQ™ Plus Mycoplasma Detection Kit

USER GUIDE

for use with:

PrepSEQ™ 1‑2‑3 Nucleic Acid Extraction Kit

PrepSEQ™ Express Nucleic Acid Extraction Kit

AccuSEQ™ Real‑Time PCR Software 3.2 or later

AccuSEQ™ Real‑Time PCR Software 2.2 or later

Catalog NumbersA55124, A57925, A57926

Publication Number MAN0028695

Revision A.0

Life Technologies Ltd | 7 Kingsland Grange | Woolston, Warrington WA1 4SR | United Kingdom

For descriptions of symbols on product labels or product documents, go to thermoﬁsher.com/symbols-deﬁnition.

Revision history:MAN0028695 A.0 (English)

Revision Date Description

A.0 25 July 2023 New document for MycoSEQ™ Plus Mycoplasma Detection Kit.

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

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. 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.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these

products, you accept the terms and conditions of all applicable Limited Use Label Licenses.

TRADEMARKS: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed.

Contents

■CHAPTER1Productinformation .................................................. 7

Productdescription ............................................................. 7

Contents and storage ............................................................ 8

Required materials notsupplied ................................................. 10

Dyes in the kit and theiruse ..................................................... 12

Testing options andapplications ................................................. 13

Instrument calibration .......................................................... 13

Workﬂow..................................................................... 14

■CHAPTER2Guidelines for sample preparation and PCR .................... 15

Guidelines for preventing contamination .......................................... 15

Guidelines for preparing sample lysates that contain targetDNA ..................... 16

Extraction controlguidelines ..................................................... 16

■CHAPTER3Manual sample extraction with the PrepSEQ™ 1‑2‑3

Nucleic Acid ExtractionKit ....................................................... 18

Prepare reagents ............................................................... 18

Prepare Magnetic Particles ..................................................... 18

Guidelines for working with Magnetic Particles .................................... 18

Option 1: Lot-release method for production harvest—supernatant or spent

media only (manual extraction) ................................................. 19

Option 2: Lot-release method for production harvest—pooled supernatant and

mammalian cells (manual extraction) ........................................... 20

Prepare sample lysate for manual extraction ....................................... 21

BindDNA ..................................................................... 22

WashDNA .................................................................... 22

EluteDNA ..................................................................... 23

MycoSEQ™ Plus Mycoplasma Detection Kit User Guide 3

■CHAPTER4Automated sample extraction with the

AutoMateExpress™ Instrument and PrepSEQ™ Express Nucleic Acid

ExtractionKit ....................................................................... 24

Prepare the PrepSEQ™ Express Cartridge ........................................ 24

Option 1: Lot-release method for production harvest—supernatant or spent

media only (AutoMateExpress™ Instrument) ..................................... 25

Option 2: Lot-release method for production harvest—pooled supernatant and

mammalian cells (AutoMateExpress™ Instrument) ................................ 27

Process samples on the AutoMateExpress™Instrument ............................ 28

■CHAPTER5Set up thePCR ..................................................... 30

Set up a MycoSEQ™ Plus Mycoplasma experiment in AccuSEQ™ Real‑Time PCR

Software v3.2 or later ........................................................ 30

Set up a MycoSEQ™ Plus or MycoSEQ™ Plus deltaRn experiment in AccuSEQ™

Real‑Time PCR Software v2.2 or later .......................................... 34

Prepare the PCR mastermix .................................................... 36

Prepare the positive template control (PTC) ....................................... 37

Prepare the PCR plate .......................................................... 37

■CHAPTER6Run the PCR and review the results—QuantStudio™ 5

Real‑Time PCR System ........................................................... 38

Load the plate in theinstrument .................................................. 39

Start therun ................................................................... 40

Monitor therun ................................................................ 41

Review MycoSEQ™ Plus results .................................................. 42

View the results for MycoSEQ™ Plus Mycoplasma experiments ................. 42

Evaluate the AmpliﬁcationPlot .............................................. 45

View and evaluate the multicomponentplot ................................... 47

Review the ﬂags in the QC summary ......................................... 48

Manually edit unknown wellcalls ............................................ 49

Acceptance criteria—AccuSEQ™ Real‑Time PCR Software v3.2 or later ............... 50

Example results with AccuSEQ™ Software v3.2 or later ............................. 52

Print reports and export results .................................................. 56

Create and print a report .................................................... 56

Export results ............................................................. 57

Contents

4MycoSEQ™ Plus Mycoplasma Detection Kit User Guide

■CHAPTER7Run the PCR and review the results—7500 Fast

Real‑Time PCRInstrument ....................................................... 58

Load the plate in theinstrument .................................................. 58

Start the instrumentrun ......................................................... 59

Review MycoSEQ™ Plus results .................................................. 60

Review the call summary ................................................... 60

View the ampliﬁcationplot .................................................. 62

View the multicomponentplot ............................................... 64

Manually edit unknown wellcalls ............................................ 66

Acceptance criteria—AccuSEQ™ Real‑Time PCR Software v2.2 or later ............... 68

Example results with AccuSEQ™ Software v2.2 or later ............................. 70

■APPENDIXATroubleshooting .................................................... 74

Troubleshooting the AccuSEQ™ software .......................................... 74

Troubleshooting the MycoSEQ™ Plus Mycoplasma DetectionKit ..................... 76

Troubleshooting speciﬁc sample types ........................................... 76

■APPENDIXBIn-process sample testing ........................................ 77

In-process testing (manual extraction) ............................................ 77

In-process testing (AutoMateExpress™ Instrument) ................................. 78

■APPENDIXCAutomated sample extraction with the KingFisher™ Flex

Puriﬁcation System ................................................................ 79

Plates needed for automated extraction using the KingFisher™ Flex

Puriﬁcation System ........................................................... 79

Prepare reagents ............................................................... 79

Prepare Magnetic Particles ..................................................... 80

Option 1: Lot-release method for production harvest—supernatant or spent

media only (KingFisher™ Flex Puriﬁcation System) ................................ 80

Option 2: Lot-release method for production harvest—pooled supernatant and

mammalian cells (KingFisher™ Flex Puriﬁcation System) ........................... 81

Set up the KingFisher™ Flex Puriﬁcation System ................................... 82

Prepare the plates .............................................................. 82

Process samples on theinstrument .............................................. 83

Contents

MycoSEQ™ Plus Mycoplasma Detection Kit User Guide 5

■APPENDIXDCreate a MycoSEQ™ Plus Mycoplasma deltaRn

experiment—AccuSEQ™ Real‑Time PCR Softwarev3.2 ...................... 85

■APPENDIXESupplemental procedures ......................................... 90

Seal the plates ................................................................. 90

Supplemental software procedures (AccuSEQ™ Real‑Time PCR Software v3.2 or later) . 91

Open an experiment ....................................................... 91

Deﬁne and assign plate and well attributes .................................... 91

Results screen ............................................................ 97

Supplemental software procedures (AccuSEQ™ Real‑Time PCR Software v2.2 or later) . 98

Add and removesamples ................................................... 98

Deﬁnesamples ............................................................ 98

Assign targets towells ..................................................... 99

Assign a sample to a well .................................................. 99

Select wells using the platelayout .......................................... 100

Select wells using the welltable ............................................ 100

Set upunknowns ......................................................... 101

Set up negative controls .................................................. 101

Edit Plot Properties ....................................................... 101

ReviewPlots ............................................................. 101

View the raw dataplot ..................................................... 102

■APPENDIXFAcceptance criteria ............................................... 104

Threshold cycle value (Ct)...................................................... 104

Results interpretation using the acceptance criteria ............................... 105

Other acceptance criteria considerations ........................................ 105

■APPENDIXGBackgroundinformation ......................................... 106

■APPENDIXHKit speciﬁcity andsensitivity .................................... 107

Sensitivity .................................................................... 107

Kitspeciﬁcity ................................................................. 107

Inclusivity—Detectablespecies ................................................. 107

Exclusivity—Undetectable organisms ............................................ 108

■APPENDIXIDocumentation and support ..................................... 110

Relateddocumentation ........................................................ 110

Customer and technical support ................................................ 110

Limited product warranty ...................................................... 111

Contents

6MycoSEQ™ Plus Mycoplasma Detection Kit User Guide

Product information

IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix

in this document.

Product description

The MycoSEQ™ Plus Mycoplasma Detection Kit is a TaqMan™-based quantitative PCR (qPCR) kit for

the detection of mycoplasma in complex bioproduction samples. Designed and tested using criteria

for rapid mycoplasma detection in biotherapeutic manufacturing cell culture lots, the kit meets or

exceeds sensitivity and speciﬁcity guidance provided in European Pharmacopoeia (E.P.2.6.7, 2007),

U.S. Pharmacopoeia (US63), and Japanese Pharmacopoeia (JP18). When used with a suitable sample

preparation method, the kit can detect less than 10CFU/mL or the genomic equivalent of 10GC/mL.

This multiplexed assay enables simultaneous screening of over 200species of mycoplasma

(determined by in silico analysis), with no demonstrated cross-reactivity to closely related bacterial

species. An internal control is included to ensure consistent PCR performance across reactions and is

used to monitor PCR inhibition. An external assay-speciﬁc control template is provided for use as a

positive plate control and sample extraction control. This control template generates a unique signal to

distinguish a contamination event from a true positive sample call.

The MycoSEQ™ Plus Mycoplasma Detection Kit is part of an integrated workﬂow for adventitious

agent, impurity, and contaminant testing during biopharmaceutical manufacturing. PrepSEQ™ nucleic

acid extraction kits enable manual or automated sample preparation, for a sample-to-result workﬂow in

under 5 hours. (Complex matrices may require additional upfront processing steps.) Kit performance

has been tested and conﬁrmed on the QuantStudio™ 5 Real‑Time PCR System and 7500 Fast

Real‑Time PCR Instrument. Data analysis using AccuSEQ™ Real‑Time PCR Software provides accurate

quantiﬁcation and security, audit, and e-signature capabilities to enable 21CFR Pt11 compliance.

The kit can be purchased separately or with one of the following PrepSEQ™ nucleic acid extraction kits

for the isolation of mycoplasma DNA:

•PrepSEQ™ 1‑2‑3 Nucleic Acid Extraction Kit

•PrepSEQ™ Express Nucleic Acid Extraction Kit

The MycoSEQ™ Plus Mycoplasma Detection Kit uses predeﬁned experiment templates that are

included with the following versions of AccuSEQ™ Real‑Time PCR Software for PCR set up, operation,

and post-run analysis:

•AccuSEQ™ Real‑Time PCR Software 3.2 or later (used with QuantStudio™ 5 Real‑Time PCR

System)

•AccuSEQ™ Real‑Time PCR Software 2.2 or later (used with 7500 Fast Real‑Time PCR Instrument)

1

MycoSEQ™ Plus Mycoplasma Detection Kit User Guide 7

Contents and storage

The MycoSEQ™ Plus Mycoplasma Detection Kit (Cat. No. A55124) is sold separately or with each of the

following kits:

•MycoSEQ™ Plus Mycoplasma Detection Kit with PrepSEQ™ 1‑2‑3 Nucleic Acid Extraction Kit (Cat.

No. A57925)—For manual sample preparation or sample preparation using the KingFisher™ Flex

Puriﬁcation System with 96 Deep-Well Head.

•MycoSEQ™ Plus Mycoplasma Detection Kit with PrepSEQ™ Express Nucleic Acid Extraction Kit

(Cat. No. A57926)—For automated sample preparation with the AutoMateExpress™ Instrument.

Kit components are listed below.

Table1MycoSEQ™ Plus Mycoplasma Detection Kit (included with all catalog numbers)

Contents Cap color Amount [1] Storage

MycoSEQ™ Plus qPCR 10X Assay Mix

green

300µL –25°C to –15°C

Negative Control

white

1,000µL –25°C to –15°C until ﬁrst thawed,

then store at 2–8°C

2x qPCR Master Mix Plus

blue

2×750µL –25°C to –15°C

MycoSEQ™ Plus Discriminatory

Positive/ Extraction Control

(1,000copies/µL) red

700µL –25°C to –15°C

DNA Dilution Buer

clear

7 mL –25°C to –15°C until ﬁrst thawed,

then store at 2–8°C

[1] The kit contains reagents for 100reactions.

Table2PrepSEQ™ 1‑2‑3 Nucleic Acid Extraction Kit (included with Cat. No. A57925)

Contents Amount[1] Storage[2]

RNase Cocktail™ Enzyme Mix[3] 2 × 1.0 mL –25°C to –15°C

Box 1

PrepSEQ™ Lysis Buer 2 × 50 mL

18–25°C (room temperature)

Binding Solution Bottle (Isopropanol)

Note: Add 30 mL of 100% isopropanol to the empty bottle

before ﬁrst use, then label the bottle to indicate that it

contains isopropanol.

1 empty bottle

Chapter1Product information

Contents and storage

1

8MycoSEQ™ Plus Mycoplasma Detection Kit User Guide

Table 2 PrepSEQ 1‑2‑3 Nucleic Acid Extraction Kit (included with Cat. No. A57925)(continued)

Contents Amount[1] Storage[2]

Wash Buer Concentrate

Note: Add 74 mL of 95% non-denatured ethanol before ﬁrst

use, then label the bottle to indicate that it contains ethanol.

18–25°C (room temperature)

2 × 26 mL

Elution Buer 25 mL

Proteinase K Buer[4] 50 mL

Proteinase K Buer II[4] 50 mL

Box 2

Magnetic Particles 2 × 1.5 mL 18–25°C (room temperature)

Box 3

Proteinase K, 20 mg/mL 1.25 mL –25°C to –15°C

[1] Contains reagents for 100 small-scale (100 μL) cell culture extractions.

[2] See the product label for the expiration date.

[3] Not required for standard mycoplasma extraction, but may be needed for high cell-density samples.

[4] Not required for mycoplasma extraction.

Table3 PrepSEQ™ Express Nucleic Acid Extraction Kit (included with Cat. No. A57926)

Contents Amount Storage

PrepSEQ™ Express Cartridge 1 box of 52 18–25°C (room temperature),

foil‑sideup

Lysis Tube 1 pack of 52 18–25°C (room temperature)

PrepSEQ™ Express Sample Tube 1 pack of 52 1.5-mL tubes and

screwcaps

Elution Tube 1 pack of 52 hinged-cap tubes

AutoMateExpress™ Tips and Tip

Holders

1 pack of 52 sets

Chapter1Product information

Contents and storage 1

MycoSEQ™ Plus Mycoplasma Detection Kit User Guide 9

Required materials not supplied

Unless otherwise indicated, all materials are available through thermoﬁsher.com. "MLS" indicates that

the material is available from ﬁsherscientiﬁc.com or another major laboratory supplier.

Item Source

Equipment

QuantStudio™ 5 Real‑Time PCR System with AccuSEQ™ Software v3.2

or later

or

7500 Fast Real‑Time PCR Instrument with AccuSEQ™ Real‑Time PCR

Software v2.2 or later

Contact local sales

representative

Vortex mixer MLS

Microtube centrifuge MLS

Plate centrifuge MLS

Centrifuge for 50-mL tubes (max. speed ≥16,000 × g)MLS

Heat blocks for 2‑mL tubes MLS

Vortex Adapter‑60 AM10014

16-position magnetic stand (DynaMag™‑2Magnet or equivalent) 12321D or equivalent

Equipment for automated DNA extraction

AutoMateExpress™ Instrument (for use with the PrepSEQ™ Express

Nucleic Acid Extraction Kit)

4441763

4467754

A38075

KingFisher™ Flex Puriﬁcation System with 96 Deep-Well Head (for

use with the PrepSEQ™ 1‑2‑3 Nucleic Acid Extraction Kit, automated

workﬂow)

5400630

(Optional) Magnetic Stand-96 (for use with the KingFisher™ Flex

Puriﬁcation System with 96 Deep-Well Head)

AM10027

Generic consumables

Disposable gloves MLS

Aerosol-resistant pipette tips MLS

Chapter1Product information

Required materials not supplied

1

10 MycoSEQ™ Plus Mycoplasma Detection Kit User Guide

(continued)

Item Source

Pipettors:

•Positive-displacement

•Air-displacement

•Multichannel

•Repeat (1µL–1mL)

MLS

2.0-mL Safe-Lock Tubes, round bottom VWR 62111-754

50-mL conical tubes MLS

RNase-free Microfuge Tubes (1.5 mL) AM12450

0.5 M EDTA MLS

Phosphate buered saline (PBS) (for the negative extraction control) MLS

RNase AWAY™ Surface Decontaminant or equivalent 7003PK (other sizes also

available)

Consumables for manual sample extraction

95% ethanol MLS

>99% isopropanol MLS

Consumables for testing supernatant or spent media only (Option 1)

PrepSEQ™ Lysis Buer (included with the PrepSEQ™ 1‑2‑3 Nucleic Acid

Extraction Kit)

A29825

PrepSEQ™ 1‑2‑3 Nucleic Acid Extraction Kit (included as part of Cat. No.

A57925)

4452222

Consumables for testing pooled supernatant/spent media plus mammalian cells (Option 2)

Cell Fractionation Buer (included with PrepSEQ™ 1-2-3 Mycoplasma

Nucleic Acid Extraction Kit)

4403461

Optional: PrepSEQ™ 1-2-3 Mycoplasma Nucleic Acid Extraction Kit

(includes Cell Fractionation Buer for pooled samples)[1]

4443789

Consumables for use with the KingFisher™ Flex Puriﬁcation System with 96 Deep-Well Head

KingFisher™ Flex 96 Deep-Well Heating Block 24075430

Pharma KingFisher™ Flex 96 Deep-Well Plates A43075

Pharma KingFisher™ Flex 96 Deep-Well Tip Combs A43074

For use with the tip comb: Pharma KingFisher™ Flex 96 Standard Plate A43076

Chapter1Product information

Required materials not supplied 1

MycoSEQ™ Plus Mycoplasma Detection Kit User Guide 11

(continued)

Item Source

Consumables for use with real-time PCR instruments

MicroAmp™ Fast Optical 96‑Well Reaction Plate with Barcode, 0.1 mL 4346906

MicroAmp™ Optical Adhesive Film 4360954 (25 covers)

4311971 (100 covers)

MicroAmp™ Adhesive Film Applicator 4333183

MicroAmp™ 96-Well Base N8010531

[1] Can also use the PrepSEQ™ 1‑2‑3 Nucleic Acid Extraction Kit plus Cell Fractionation Buffer.

Dyes in the kit and their use

Dye Detected in Use

FAM™Test samples that contain

mycoplasma

Positive template controls

and positive extraction

controls

Detects the presence of mycoplasma in test samples, and

veriﬁes ampliﬁcation of the mycoplasma target sequence in

positive controls.

VIC™Positive template controls

and positive extraction

controls

Test samples and negative

controls that have been

contaminated by the positive

control

Detects contamination of test samples or negative controls

by the positive control.

The MycoSEQ™ Plus Discriminatory Positive/ Extraction

Control includes a speciﬁc sequence whose presence is

indicated by the VIC™ signal during PCR ampliﬁcation.

NED™All samples and controls

(positive and negative)

Detects PCR inhibition. If there is a large amount of host-cell

DNA, the NED™ signal (used as an internal positive control)

can be inhibited or absent in test samples. Test samples with

host-cell DNA competition show the following:

•No target-speciﬁc signal (FAM™ dye)

•Ct >38.00 or undetermined for the IPC signal (NED™

dye)

•No positive control signal (VIC™ dye)

If there is PCR inhibition and the target-speciﬁc signal (FAM™

dye) is present, the IPC signal (NED™ dye) can be ignored

and the sample considered positive for the presence of

mycoplasma.

ROX™Not reported by the software Used as a passive reference dye by the instrument.

Chapter1Product information

Dyes in the kit and their use

1

12 MycoSEQ™ Plus Mycoplasma Detection Kit User Guide

Testing options and applications

This user guide provides two main testing options for lot-release applications (Option1 and 2), plus an

option for in-process testing. Option 1 and 2 test procedures have been conﬁrmed during development

to meet release-testing sensitivity requirements. However, customers should qualify and validate these

methods for their own products, workﬂows, and applications.

Application

Testing options

Option 1: Testing of

supernatant or spent

media only

Option 2: Testing of

pooled supernatant/media plus

mammalian cells

In-process

testing

Cell therapy lot release

<1×106 cells/sample

>1×106 cells/sample



Monoclonal antibody (mAb)

lot release



Other therapeutic lot

release

<1×106 cells/sample

>1×106 cells/sample



Cell banks  

Plasmids  

Raw materials  

Buers and media  

Final drug product  

Surveillance  

Bioreactor expansion  

Bioreactor "quick release"

to puriﬁcation (pending

compendial test)

 

Instrument calibration

Before using the MycoSEQ™ Plus Mycoplasma Detection Kit on the following instruments, you must

calibrate each instrument for FAM™, VIC™, ROX™, and NED™ dyes:

•QuantStudio™ 5 Real‑Time PCR System—For calibration instructions, see the QuantStudio™ 3 and

5 Real‑Time PCR Systems Installation, Use, and Maintenance Guide (Pub. No. MAN0010407).

•7500 Fast Real‑Time PCR Instrument—For calibration instructions, see the Applied Biosystems™

7500/7500 Fast Real‐Time PCR System: Maintenance Guide (Pub. No. 4387777).

Chapter1Product information

Testing options and applications 1

MycoSEQ™ Plus Mycoplasma Detection Kit User Guide 13

Workﬂow

Manual sample extraction—PrepSEQ™ 1‑2‑3 Nucleic Acid Extraction Kit

(page18)

OR

Automated sample extraction—AutoMateExpress™ Instrument (page24)

OR

Automated sample extraction—KingFisher™ Flex Puriﬁcation System with

96 Deep-Well Head (page79)

Prepare the PCR plate (page30)

Run the PCR and review the results—QuantStudio™ 5 Real‑Time

PCR System (page38)

OR

Run the PCR and review the results—7500 Fast Real‑Time

PCR Instrument (page58)

Chapter1Product information

Workﬂow

1

14 MycoSEQ™ Plus Mycoplasma Detection Kit User Guide

Guidelines for sample preparation

and PCR

Guidelines for preventing contamination

•Store kit reagents in a separate location from DNA samples.

•Use separate, clean, designated work areas and equipment (preferably separate PCR hoods) for

preparing the DNA template and preparing the PCR master mix, to minimize cross-contamination.

•Do not bring ampliﬁed products into the reaction setup area.

•Wear clean gloves, a clean laboratory coat, and protective eyewear.

•Do not reuse gloves and coats between ampliﬁcation and preparation reactions.

•Change gloves if you suspect that they are contaminated.

•Use a positive-displacement pipettor or aerosol‑resistant barrier pipette tips.

•Use low-retention or siliconized microtubes when preparing unknown samples.

•Open and close all sample tubes carefully. Avoid splashing or spraying samples.

•Keep reactions and components capped as much as possible.

•Always include a no-template control (NTC) reaction to detect contamination.

•Do not unseal reaction plates after ampliﬁcation is complete. Unsealing the plates increases the

risk of contaminating subsequent reactions with ampliﬁed product.

•Clean laboratory benches and equipment periodically with a nucleic acid decontamination solution

such as RNase AWAY™ Surface Decontaminant.

2

MycoSEQ™ Plus Mycoplasma Detection Kit User Guide 15

Guidelines for preparing sample lysates that contain target

DNA

Minimizing cellular DNA and/or RNA in the ﬁnal extracted DNA is critical to mycoplasma DNA detection.

High amounts of cellular DNA and/or RNA can cause PCR inhibition and high background, reducing

detection of low-copy targets. Factors that aect levels of cellular DNA and/or RNA include:

•Viability of cell culture sample—Use fresh culture samples to increase the purity of the extracted

target DNA. Avoid conditions such as long-term storage (>4 days) at 4°C or freezing temperatures.

Such temperatures cause increased death or lysis of cells, which contributes to additional

background DNA in samples.

•In lot-release protocols, when processing the mammalian cell pellet, keep the pellet on ice

and perform all processing steps at 4°C to minimize host cell nuclei lysis. Processing at room

temperature increases lysis of nuclei and host DNA in the ﬁnal extracted DNA and causes reduced

extraction eciency.

•In lot-release protocols with pooled supernatant and mammalian cells:

–Use Cell Fractionation Buer to limit host-cell nuclei lysis. The buer lyses mycoplasma cell

membranes but leaves host-cell nuclei intact.

–In some cases, the cell pellet is large and sticky and cannot be resuspended easily. Never

vortex to resuspend the cells.

–When transferring the cell culture supernatant, avoid touching the pellet, which contains nuclei

and viscous material that may be generated from lysis of nuclei. If needed, use a P200 pipette

to perform the transfer.

–In the ﬁnal transfer of the cell pellet supernatant, avoid contact with or transfer of the viscous

material. If needed, recentrifuge the tube at 1000×g for 3 minutes at 4°C, then very carefully

transfer 300 µL (two 150‑μL aliquots) with a P200 pipette.

Extraction control guidelines

The MycoSEQ™ Plus Discriminatory Positive/ Extraction Control provided in the kit is a multi-purpose

control that can be used as both a positive extraction control (PEC) and a positive template control

(PTC).

If you are running a PEC with each sample, we recommend that you extract and analyze 1 replicate of

each test sample without control and 1 replicate with control:

Type of control Description Number to run Purpose

Positive

extraction control

(PEC)

MycoSEQ™ Plus

Discriminatory Positive/

Extraction Control (2,000–

3,000 copies) in 1X PBS (free

of Mg and Ca)

1 per extraction run •Verify reagent and system

performance.

•Evaluate the eciency

of DNA extraction and

detection from test

samples.

Chapter2Guidelines for sample preparation and PCR

Guidelines for preparing sample lysates that contain target DNA

2

16 MycoSEQ™ Plus Mycoplasma Detection Kit User Guide

(continued)

Type of control Description Number to run Purpose

Negative

extraction control

(NEC)[1]

1X PBS (free of Mg and Ca) 1 per extraction run Monitor for contamination

of the extraction reagents,

equipment, and work areas.

Positive

extraction control

plus sample (S-

PEC)

MycoSEQ™ Plus

Discriminatory Positive/

Extraction Control (2,000–

3,000 copies) plus test sample

1 per sample:

•Sample 1, tube 1

—Test sample

•Sample 1, tube 2

—Positive control

plus test sample

Evaluate the eciency of DNA

extraction and detection from

test samples.

QC sample used

as a negative

extraction control

(S-NEC)[1]

Sample that is known to not

contain mycoplasma

1 per extraction run Monitor for contamination

of the extraction reagents,

equipment, and work area.

Internal positive

control (IPC)

An artiﬁcial sequence in the

MycoSEQ™ Plus qPCR 10X

Assay Mix that generates a

NED™ dye signal.

Included in every

reaction.

Monitor for PCR inhibition.

[1] Optional during routine testing.

Chapter2Guidelines for sample preparation and PCR

Extraction control guidelines 2

MycoSEQ™ Plus Mycoplasma Detection Kit User Guide 17

Manual sample extraction with

the PrepSEQ™ 1‑2‑3 Nucleic Acid

Extraction Kit

Prepare reagents

Before ﬁrst use of the PrepSEQ™ 1‑2‑3 Nucleic Acid Extraction Kit, prepare the following reagents:

•Binding Solution—Add 30 mL of 100% isopropanol to the empty Binding Solution bottle. Mark the

bottle label to indicate that isopropanol has been added.

•Wash Buer—Add 74 mL of 95% non-denatured ethanol to the Wash Buer Concentrate bottle,

then mix well. Mark the bottle label to indicate that ethanol has been added.

Prepare Magnetic Particles

Before each use, incubate the Magnetic Particles at 37°C for at least 10 minutes with intermittent

vortexing at setting #7 or until the particles are completely suspended.

Note: Extended heating and vortexing may be required for hard-to-resuspend beads.

Guidelines for working with Magnetic Particles

•When placing sample tubes into the magnetic stand, always orient the Magnetic Particles pellet

toward the magnet.

•Except where noted, DNA capture is complete after ~1 minute in the magnetic stand.

•When separating the liquid phase or eluate from the Magnetic Particles, do not disturb the

particles. Carryover of particles can inhibit PCR.

•During wash steps, it is not necessary to detach the particles from the tube wall. Particle adherence

to the tube wall does not aect DNA recovery. Some samples cause the particles to adhere very

ﬁrmly to the tube wall. For other samples, the particles form loose aggregates that readily detach

during vortexing. The particles must disperse into a slurry during heating and vortexing in the

elution step of the protocol.

3

18 MycoSEQ™ Plus Mycoplasma Detection Kit User Guide

Option 1: Lot-release method for production harvest—

supernatant or spent media only (manual extraction)

Supernatant

Lysate

Mycoplasma pellet

Centrifuge at high speed

• Centrifuge the sample

• Transfer the supernatant to another tube

DNA extraction

Lysis

(skip for cell therapy applications)

Follow this procedure to test supernatant, spent media, and waste stream samples. This high-sensitivity

protocol (LOD≤ 10 CFU/mL) is suggested for lot-release testing. Use this procedure for cell therapy

applications and other production harvest processes where no cells or <1×106 cells are tested.

If you are running a positive extraction control for each sample (S-PEC), prepare 2 tubes for each

sample: one to be spiked with positive control and one without.

For each sample, perform the following steps:

1. (For cell therapy applications only) Add 200,000 cells to 10mL of sample in a 50‑mL conical tube,

then proceed to step4.

2. Add 10 mL of sample to a 50‑mL conical tube, then centrifuge at 1,000×g for 5 minutes at 4°C.

3. Transfer the supernatant to a new 50‑mL conical tube without disturbing the mammalian cell pellet,

then place the supernatant on ice. Discard the pellet.

4. Centrifuge the tube at 16,000×g for 30 minutes at 4°C to collect the mycoplasma.

5. Carefully remove and discard the supernatant without touching or disturbing mycoplasma pellet.

Use a pipette to carefully remove any remaining liquid.

6. Add 300μL of PrepSEQ™ Lysis Buer to the mycoplasma pellet. Gently vortex for 5 seconds to

completely resuspend the pellet. If the pellet is dicult to dislodge, vigorously agitate the tube.

Proceed to “Prepare sample lysate for manual extraction” on page21.

Chapter3Manual sample extraction with the PrepSEQ™ 1‑2‑3 Nucleic Acid Extraction Kit

Option 1: Lot-release method for production harvest—supernatant or spent media only (manual extraction) 3

MycoSEQ™ Plus Mycoplasma Detection Kit User Guide 19

Option 2: Lot-release method for production harvest

—pooled supernatant and mammalian cells (manual

extraction)

Supernatant with

released from cells

Mycoplasma pellet

Mycoplasma

Cell pellet Supernatant

Centrifugation

Differential cell lysis

Low-speed

centrifugation

DNA extraction

Lysis

High-speed

centrifugation

Lysate

Follow this procedure to test supernatant samples pooled with mammalian cell lysate. This high-

sensitivity protocol (LOD≤ 10 CFU/mL) is suggested for lot-release testing. Use this procedure for

monoclonal antibody applications and other production harvest processes where >1×106 cells are

tested.

If you are running a positive extraction control for each sample (S-PEC), prepare 2tubes for each

sample: one spiked with positive control and one without.

For each sample, perform the following steps:

1. Add 10 mL of sample to a 50‑mL conical tube, then centrifuge at 1,000×g for 5 minutes at 4°C.

2. Transfer the supernatant to a new 50‑mL conical tube without disturbing the mammalian cell pellet,

then place the supernatant on ice.

3. Remove residual supernatant from the pellet. Keep the pellet on ice for use in step6.

4. Centrifuge the supernatant (from step2) at 16,000×g for 30 minutes at 4°C to collect the

mycoplasma.

5. Carefully remove and discard the supernatant without touching or disturbing the mycoplasma

pellet. Use a pipette to carefully remove any remaining liquid. Retain the mycoplasma pellet for use

in step9.

Chapter3Manual sample extraction with the PrepSEQ™ 1‑2‑3 Nucleic Acid Extraction Kit

Option 2: Lot-release method for production harvest—pooled supernatant and mammalian cells (manual extraction)

3

20 MycoSEQ™ Plus Mycoplasma Detection Kit User Guide

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Thermo Fisher Scientific MycoSEQ Plus Mycoplasma User guide

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