APPLICATION NOTETaqMan® Pathogen Assays

Viral pathogen monitoring in human cell culturesTaqMan® Gene Expression Assays for Pathogen Research

New TaqMan® Gene Expression Assays and TaqMan® Array Card for human viral pathogen researchThe ability to work with human cells in vitro has been a critical tool in our quest to better understand human disease. It facilitates investigation of cellular processes for basic research programs, and also has profound implications in the field of regenera-tive medicine. Central to this newly evolving field are stem cells—plu-ripotent cells capable of indefinite self-renewal and, when conditions are optimal, giving rise to a wide range of mature cell types. Scientific advances in culturing and controlling stem cell characteristics significantly expand the repertoire of cell-culture models for development and human disease research, and have the potential to eventually spawn therapies for treat-ing injuries and diseases such as Alzheimer’s, Parkinson’s, and diabetes mellitus.

With the increased ability to manipulate the cellular environment, however, comes an increased risk of environmental contamination. Cell cultures are subject to potential contamination from a wide variety of pathogens including viruses, Mycoplasma, bacteria, and fungi through exposure to the lab environ-ment, personnel, and contaminated reagents. These risks are perhaps most critical when culturing cells for research. Here, we introduce the use of novel TaqMan® Gene Expression Assays—PCR primer/TaqMan® probe

sets—for real-time PCR screening and detection of common viral pathogens in research cell lines. As part of their development, we rigorously tested each assay for sensitivity, dynamic range, and amplification efficiency. We then assembled the assays on 384-well TaqMan® Array Cards and retested in this easy-to-use microfluidic format. Here, we present the results of these analyses, which demonstrate that this quantitative PCR (qPCR) approach for viral patho-gen monitoring of human cell lines for research uses a simple, fast method to deliver highly sensitive and reproducible results.

In silico assay designThe PCR primers and TaqMan® probe that constitute TaqMan® Assays for Pathogen Detection are designed using a proprietary algorithm that maximizes the user’s ability to detect viral sequences and discriminate between true-target and nontarget sequences, even when they are highly related. The algorithm considers the effects of primer melting temperatures, physical-chemical thermodynamic parameters, and potential primer-dimer formation. Candidate assays are also mapped to nearest-neighbor and nontarget sequences to adjust primer and probe design for maximum target specificity.

Assay designs derived from this process are highly sensitive, highly specific, and share highly similar thermodynamic properties, enabling them to be run under identical thermal cycling conditions—an important consideration when placing them together on 384-well TaqMan® Array Microfluidic Cards.

In most cases (e.g., human herpesvirus 5, CMV), a single TaqMan® Assay is sufficient to detect the target virus; however, in some cases (e.g., human immune deficiency, HIV), two or more TaqMan® Assays are used in multiplex reactions to detect all sequence variants associated with these highly polymorphic viruses. To design multiplex assay mixtures, the algorithm selected the fewest individual assays needed to detect all forms of the virus, while avoiding assays with pre-dicted sequence interactions, to help ensure that the assays would work well in multiplex. Available TaqMan® Assays for Pathogen Detection are shown in Table 1.

More detailed information about all GenBank accession numbers detected by these assays can be found online at http://www3.appliedbiosystems.com/cms/groups/portal/documents/generaldocuments/cms_070848.xls.

Organism Assay ID Gene symbol GenBank accession Notes

Human papillomavirus type 16 HPV16 Pa03453396_s1 E1 K02718

Human herpes simplex virus 1 HSV-1 Pa04230116_s1 UL41 AB009267

Human herpesvirus 4 HHV-4, EBV Pa03453399_s1 IR1 AJ507799.2

Human herpesvirus 5 HHV-5, CMV Pa03453400_s1 UL132 AY446894

Human herpesvirus 7 HHV-7 Pa03453404_s1 U48 AF037218

BK polyomavirus BKV Pa03453401_s1 VP2 V01108

JC polyomavirus JCV Pa03453402_s1 VP2 J02226

Simian virus 40 SV-40 Pa03453403_s1 VP2 J02400

Hepatitis C virus HCV Pa03453408_s1 5' UTR AF009606

Hepatitis B virus HBV Pa03453405_s1Pa03453406_s1

P, SP, X

X04615X04615

Orangutan hepadnavirus and duck hepatitis B virus are also detected.

Human T-lymphotropic virus 1 HTLV-I Pa03453397_s1 rex, tax AF033817

Human T-lymphotropic virus 2 HTLV-II Pa03453398_s1Pa03453407_s1

LTRLTR

M10060L11456

Human immunodeficiency virus 1 HIV-1 Pa03453409_s1 Pa03453410_s1

LTRLTR

K03455AF196760

Chimpanzee SIV (SIVcpz) is also detected. The relatively rare group N and O HIV-1 viruses are not detected.

Human immunodeficiency virus 2 HIV-2 Pa03453414_s1Pa03453415_s1

LTRLTR

M33262J04498

Sooty mangabey SIV (SIVsm) and macaque SIV (SIVmac) are also detected.

Human adenovirus 2Human adenovirus 5Human adenovirus 1Human adenovirus C

HAdV-CPa03453411_s1Pa03453412_s1Pa03453413_s1

L4L3L4

J01917AY601635AF534906

Some strains of human adenovirus 6 are detected.Feline adenovirus is also detected.

Table 1. TaqMan® Gene Expression Assays for Pathogen Detection.

Methods and experimental designBecause TaqMan® Assays for Pathogen Detection are available in either single tubes or preconfigured and ready to use on the TaqMan® Array Human Viral Pathogen Screening Card, performance was verified in both formats. Synthetic DNA targets, biological standards (when avail-able), and nucleic acids from positive-control cell lines were used for these validation experiments. Nucleic acids isolated from biological standards and positive controls from other sources were quantified by converting spec-trophotometer readings using the extinction coefficient for RNA, and dilution series of nucleic acid were used for PCR reactions. Synthetic DNA targets were serially diluted and reactions included 1–105 copies in 10-fold increments. PCR was conducted with a one-step real-time RT-PCR strategy using Ambion® AgPath-ID™ One Step RT-PCR Reagents (e.g., Cat. No. 4387424M). Samples were reverse transcribed for 10 min at 45°C, then amplified for 40 cycles. For single-tube assays, the thermal cycle was 95°C, 15 sec denaturation followed by 60°C, 45 sec elongation. For the TaqMan® Array Cards, the thermal cycle was 97°C for 15 sec followed by 59.7°C for 45 sec. Triplicate reactions were run and detected on Applied Biosystems® real-time PCR instruments.

Assays exhibit single-copy detection, 5-log dynamic range, and 100% (±10%) amplification efficiencyAssay validation using synthetic targetsOligonucleotides corresponding to the viral sequences targeted by TaqMan® Assays for Pathogen Detection were synthesized to serve as templates in validation experi-ments. These synthetic viral templates were serially diluted and amplified in triplicate to construct a 10-fold, 6-point standard curve that also included no-template controls (NTCs). In these experiments, all of the TaqMan® Assays succeeded in detecting a single copy of synthetic target with Ct values of 33.5–37.5 cycles, although only some of the assays showed linear data through reactions containing just one copy of target (Figure 1). Most assays did, however, result in a straight line when plotted as a standard curve in reactions containing 10–105 copies of viral target; the remaining few showed a linear result when 102–105 copies were present. This wide dynamic range means that most samples will not require dilution or concentration before TaqMan® Assay–based testing. Amplification efficiency of the assays ranged from 92–105%; for example, the slope of the standard curve of HPV16 assay results was –3.398, which corresponds to an amplification efficiency of 96.9%. High amplification efficiency is required for relative

Virus Nucleic acid source Provider

HPV16 Propagated in SiHa cells and Ca Ski cells “HPV Low” ATCC HTB35, containing 1–2 copies of HPV-16/cell “HPV High” ATCC CRL-1550, ~600 copies of HPV-16/cell

HTLV-1 Virus strain MT-2 prepared from virus infected cells (MT-2) Advanced Biotechnologies Inc.

HHV4 (EBV) Propagated in Raji cell line ATCC CCL-86

HHV5, CMV Virus reference strain AD169 from infected MRC-5 cells Division of Virology, NIBSC

BK human polyomavirus Gel-purified viral DNA from plasmid containing entire BKV genome (MM strain) Advanced Biotechnologies Inc.

JC human polyomavirus Gel-purified viral DNA from plasmid containing entire JCV genome (MAD1 strain) Advanced Biotechnologies Inc.

SV40 Virus strain prepared from infected Cos7 cell line ATCC CRL-1651

HHV-7 human herpesvirus 7 Virus strain H7-4 purified virus Advanced Biotechnologies Inc.

HCV Hepatitis C virus RNA, 3rd WHO International Standard NIBSC

HBV Hepatitis B virus DNA 2nd WHO International Standard NIBSC

HTLV-2 Virus strain C3-44/Mo prepared from virus-infected cells (C3-44/Mo) Advanced Biotechnologies Inc.

HIV 1 HIV-1 RNA WHO 2nd International Standard NIBSC

Adenovirus C Clinical isolates amplified in tissue culture Division of Virology, NIBSC

HIV 2 HIV-2 RNA WHO 1st International Standard NIBSC

HSV1 Clinical isolates amplified in tissue culture Division of Virology, NIBSC

Table 2. Viral biological standards and known-infected cell lines used in the validation experiments.

Figure 1. TaqMan® Assays for Pathogen Detection exhibit single-copy sensitivity, 5-log dynamic range, and excellent amplification efficiency. The indicated number of copies of synthetic viral targets were amplified using the corresponding TaqMan® Assay. The real-time PCR data were plotted as standard curves; the resulting straight lines indicate that amplification signal corresponds linearly to input nucleic acid quantity.

105

10152025303540

10 102 103 104 105 106

Adenovirus C

Ct

Input copy number

y = -3.5839x + 40.196R2 = 0.99934

105

10152025303540

10 102 103 104 105 106

BKV

Ct

Input copy number

y = -3.3528x + 41.845R2 = 0.9991

105

10152025303540

10 102 103 104 105 106

EBV2

Ct

Input copy number

y = -3.3142x + 40.853R2 = 0.99952

105

10152025303540

10 102 103 104 105 106

HBV

Ct

Input copy number

y = -3.4403x + 39.222R2 = 0.99897

105

10152025303540

10 102 103 104

HCV AT

Ct

Input copy number

y = -3.56x + 37.894R2 = 0.9999

105

10152025303540

10 102 103 104 105 106

HHV7

Ct

Input copy number

y = -3.2361x + 36.988R2 = 0.99875

105

10152025303540

10 102 103 104 105 106

HPV16

Ct

Input copy number

y = -3.0465x + 40.385R2 = 0.98922

105

10152025303540

10 102 103 104 105 106

HTLV2

Ct

Input copy number

y = -3.4264x + 38.44R2 = 0.99811

105

10152025303540

10 102 103 104 105 106

JCV

Ct

Input copy number

y = -3.4147x + 40.137R2 = 0.99891

Figure 2. TaqMan® Assays for Pathogen Detection provide highly specific detection of their intended viral target. Nucleic acid samples (10 ng) from cell lines infected with HPV amplified with all assays. No amplifica-tion products were seen from any of the heterologous assays, but the HPV16 assay produced a strong signal, as expected.

Cycle

ΔRn

Amplification Plot

quantification and sensitive detec-tion. Finally, no amplification was detected in the no-template control reactions—demonstrating that the reactions did not produce nonspecific signal due to TaqMan® probe deg-radation and that they were free of contaminating nucleic acid.

Comparable validation results with biological positive controlsIn a second set of experiments, selected TaqMan® Assays for Pathogen Detection were tested using nucleic acids purified from the biologi-cal standards and positive-control cell lines listed in Table 2. These known-positive samples were diluted and used as template in a similar series of assay-validation reactions. The results were comparable to those obtained using synthetic templates. All of the assays tested showed excellent linear-ity and precision, with R2 values >0.97 and PCR efficiencies of 96–108%. In addition, all were able to detect target amounts in the single-copy range with Ct values of 34–39 cycles.

Virus Assay ID Dynamic range (orders of magnitude) Slope*

HPV (“Low”) Pa03453396_s1 5 –3.4

HPV (“High”) Pa03453396_s1 5 –3.32

HHV4 (EBV) Pa03453399_s1 4 –3.3

CMV Pa03453400_s1 5 –3.4

SV40 Pa03453403_s1 3 –3.4

HCV Pa03453408_s1 3 –3.13

HBV Pa03453405_s1 Pa03453406_s1 3 –3.4

AdenovirusPa03453411_s1 Pa03453412_s1 Pa03453413_s1

4 –3.4

HSV-1 Pa04230116_s1 5 –3.6

Table 3. TaqMan® Assay performance in validation experiments using nucleic acids from biological standards and known-infected cell lines listed in Table 2. Positive control nucleic acid was serially diluted and amplified using selected TaqMan® Assays for Pathogen Detection; the results were plotted as standard curves. Dynamic range describes the assay’s ability to produce a linear response to the stated number of orders of magnitude of input template. The slope of the straight line produced in plotting the data reflects the assay’s amplification efficiency: how close to perfectly driving the synthesis of one copy of each template molecule in each round of amplification. * A slope of –3.32 represents 100% amplification efficiency.

Specificity testingNext, the assays were tested for cross-detection of highly related and non-target viruses using nucleic acid isolated from seven biological standards and viral-pathogen positive control cell cultures. Most of the assays, such as the TaqMan® HPV16 Assay, produced a strong amplifica-tion signal (Ct = 16) from reactions containing target nucleic acid, but no detectable signal from reactions with nucleic acid from uninfected samples or controls for other viruses (Figure 2). Some assays did show negligible cross-amplification, however; signal was significantly lower (Ct >35), than that from the intended target. We found approximately 105- to 106-fold lower sensitivity for the “wrong” target.

One exception was the Adenovirus C assay, which detected Adenovirus C in five out of seven cell lines tested (Figure 3). We suspected that these results were due to contamination of these cell lines with Adenovirus C in addition to being infected with the

expected viral pathogen. The supplier of the cell lines does not test for Adenovirus C. In order to find out, we challenged the assay by attempting to amplify synthetic targets for the for HHV5 (CMV), HBV, and HCV assays using the TaqMan® Adenovirus C Assay. We found that no amplification product was seen, even in reactions that contained 10,000 copies of the “wrong” targets: synthetic HBV, HHV5 (CMV), and HCV (data not shown).

In summary, all TaqMan® Assays for Pathogen Detection demonstrated at least 105-fold higher sensitivity for their specific target relative to any of the nearest neighbors tested. This minimal cross-reactivity is considered negligible. Note also that these cross-reactivity experiments included very high levels of heterologous viral target, with nucleic acid isolated from very high-titer cell lines. The amount of viral nucleic acid in the least dilute samples was approximately three orders of magnitude higher than biologically relevant samples would likely contain. The specificity testing described here pushed the assays well beyond target levels expected to be encountered in standard practice.

Running the assays on 384-well TaqMan® Array CardsFor ease of use and robust perfor-mance, TaqMan® Assays for Pathogen Detection are available on 384-well TaqMan® Array Cards. The collection is called the TaqMan® Array Human Viral Pathogen Screening Card; its layout is shown in Figure 4. TaqMan® Array Cards offer the following benefits:

• Ready to use, right out of the box—for the viruses that require multiple assays for full coverage, assays are pre-pooled before deposition on the TaqMan® Array Card for less pipetting and simplified data analysis

• Triplicate reactions—the TaqMan® Array Card includes triplicate reactions for each viral pathogen to help ensure that you get accurate and trustworthy results

• Positive controls included—for confirmation that the PCR conditions are appropriate, triplicate reactions with a positive control TaqMan® Assay targeting 18S ribosomal RNA are included

TaqMan® Array Card validationTo compare TaqMan® Array Human Viral Pathogen Screening Card results to assays run in 96-well plates, we tested the sensitivity and specificity of the TaqMan® Array Cards using both synthetic templates and nucleic acids isolated from the biological standards and positive control cultures using a similar set of experiments to those previously described (Figure 5). These experiments, however, included more comprehensive cross-reactivity testing. Since each loading port in the TaqMan® Array Cards delivers sample to triplicate reactions for each assay, these experiments were extremely fast and easy to conduct. Loading one artificial template or nucleic acid sample per port, we could evaluate the specificity and cross-reactivity for each assay in parallel. Selected data from these experiments are shown in Figure 5A. The slopes shown here correspond to an efficiency range

Figure 3. Likely adenovirus C contamination of biological standards. The TaqMan® Adenovirus C Assay produced a positive amplification signal in samples from five of seven biological controls and positive-control cell lines for other viruses. This amplification plot shows the data obtained when all of the TaqMan® Assays for Pathogen Detection were tested for cross-reactivity using nucleic acid isolated from the HHV5 (CMV)-biological control cell line. A strong signal is seen when using the TaqMan® HHV5 (CMV) Assay (blue) and a weak signal is also seen from the amplification using the TaqMan® Adenovirus C Assay (pink).

Figure 4. TaqMan® Array Human Viral Pathogen Screening Card layout. The card allows for detection of 15 viral pathogens simultaneously plus one positive control (18S).

Easy to load and runUsing 384-well TaqMan® Array Cards is fast and simple. The microfluidic technology uses eight sample-loading ports, each connected to 48 reaction wells. Simply add PCR master mix to your eight samples, mix, then pipette into the eight sample-loading ports and briefly centrifuge. This distributes your sample plus master mix to the well of the TaqMan® Array Card, where they will mix with pre-dispensed TaqMan® Gene Expression Assays for Pathogen Detection in triplicate reactions. Within minutes, your TaqMan® Array Card is ready to run on the Applied Biosystems® ViiA™ 7 or 7900HT Fast Real-Time PCR System.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

18S rRNA HHV-5, CMV BKV

HTLV-2 HIV-1 HAdV-C

18S rRNA HHV-5, CMV BKV JJCCVV

HTLV-2 HIV-1 HAdV-C HIV-2

18S rRNA HHV-5, CMV BKV

HTLV-2 HIV-1 HAdV-C

HIV-1 HAdV-C

18S rRNA HHV-5, CMV BKV

HTLV-2 HIV-1 HAdV-C

HIV-1 HAdV-C

HHV-4, EBV 18S rRNA HHV-5, CMV BKV

HIV-1 HAdV-C HIV-2

HHV-4, EBV 18S rRNA HHV-5, CMV BKV

HBV HTLV-2

HHV-5, CMV

HIV-1

BKV

HAdV-C

HHV-4, EBV 18S rRNA HHV-5, CMV BKV

HBV

18S rRNA

HTLV-2

HBV HTLV-2

HBV HTLV-2

HCV

HCV

HTLV-I

HTLV-I

HTLV-I

HCV

HTLV-I

HTLV-I

HCV

HTLV-I

HTLV-I

HTLV-I

HCV

HCV

HCV

HCV

HHV-7

HHV-7

HPV16

HPV16

HPV16

HPV16

HPV16

HPV16

HHV-7

HHV-7

HHV-7

HHV-7

HPV16

HHV-7

HPV16

HHV-7

JJCCVV

HIV-2

HHV-4, EBV

HBV

HHV-4, EBV

JJCCVV

HIV-2

JJCCVV

HHV-4, EBV

HBV

HHV-4, EBV

HBV

HHV-4, EBV

HBV

JJCCVV

HIV-2

JJCCVV

HIV-2

JJCCVV

HIV-2

SV-40

HHSSVV--11

SV-40

HHSSVV--11

HIV-2

JJCCVV

SV-40

HHSSVV--11

SV-40

HHSSVV--11

SV-40

HHSSVV--11

SV-40

HHSSVV--11

SV-40

HHSSVV--11

SV-40

HHSSVV--11

1

2

3

4

5

6

7

8

of 97–110% and all R2 values are above 0.995. Furthermore, no cross-amplification was seen in this set of experiments when a Ct <35 was used as the cut-off for a positive signal, as is common practice.

We also evaluated the dynamic range of the assays when configured in TaqMan® Array Cards by loading a dilution series of total nucleic acid extracted from biological standards and positive control cultures (see Table 2 and Figure 5B). The results confirmed our prior findings: negli-gible cross-reactivity and no signal from no-template-control reactions, linear response to increasing template

Figure 5. TaqMan® Array Human Viral Pathogen Screening Cards show sensitive virus detection, precise results, wide dynamic range, and optimal amplification efficiency. (A) A dilution series of synthetic template was loaded into each port of the TaqMan® Array Card to generate a standard curve containing the indicated number of copies of artificial template per reaction or microliter. Buffer was loaded into the remaining port for no-template control reactions. (B) The indicated target copy numbers or mass amounts of nucleic acid purified from the biological standards and positive-control cell lines were also evaluated using the TaqMan® Array Human Viral Pathogen Screening Cards.

quantities, and excellent amplification efficiency for all assays and assay sets.

Accurate and robust viral pathogen testing in cell culturesTaqMan® Gene Expression Assays for Pathogen Detection showed excellent performance (sensitivity, specificity, and dynamic range) when using synthetic viral templates and nucleic

acid from biological standards and positive control cell cultures. Eleven of the 15 assays did not show any cross-reactivity and were highly specific for their intended targets. Four assays showed negligible cross-amplification from samples containing heterologous viral nucleic acid. This cross-signal does not affect the utility of the assays because of its extremely low level and because it was only observed with

105

10152025303540

10 102 103 104 105 106

HTLV2

Ct

Input copy number

y = -3.3916x + 38.352R2 = 0.99913

0.0010

5

10

15

20

25

30

0.10.01 1 10 100 1,000 10,000

Adenovirus C Cells

Ct

Input nucleic acid target (ng)

y = -3.2711x + 18.446R2 = 0.99222

105

10152025303540

10 102 103 104

BKV Cells

Ct

Input copy number

y = -3.7142x + 39.25R2 = 0.99823

0.105

10152025303540

1.0 10 100 1,000

HPV16 Cells

Ct

Input nucleic acid target (ng)

y = -3.3265x + 28.887R2 = 0.99354

10

5

10

15

20

25

30

35

10 102 103 104

JCV Cells

Ct

Input copy number

y = -3.0581x + 36.012R2 = 0.99197

A. Synthetic viral templates B. Viral standards

105

10152025303540

10 102 103 104 105 106

HTLV1

Ct

Input copy number

y = -3.4133x + 35.445R2 = 0.99524

105

10152025303540

10 102 103 104 105 106

HIV2

Ct

Input copy number

y = -3.09x + 34.82R2 = 0.99698

05

10152025303540

1 101 102 103 104 105 106

HCV

Ct

Input copy number

y = -3.3985x + 30.797R2 = 0.99863

References

1. Thomson J, Itskovitz-Eldor J, Shapiro S et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282 (5391):1145–1147.

2. Yu J, Vodyanik MA, Smuga-Otto K et al. (2007) Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Science 318(5858):1917–1920.

3. Takahashi K, Tanabe K, Ohnuki M et al. (2007) Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 131(5):861–872.

AcknowledgementsWe thank Lyn Healy, UK Stem Cell Bank at the National Institute for Biological Standards and Control-HPA, and Ronald P. Hart, Rutgers Stem Cell Research Center, Rutgers University, Piscataway, NJ, USA for conducting many of the experiments discussed here and for sharing data with us. We also acknowledge Jacqueline Fryer, Sarah Gilbert, Anna Gottleib, Claire Morris, and Lesley Young, National Institute for Biological Standards & Control (NIBSC-HPA) for generously providing some of the biological standards and positive-control cell lines.

In addition, we thank Kelly Warrington, Sr. Field Application Specialist, Life Technologies, for coordinating this study. This collaboration was lead by Astrid Ferlinz, FALCON Team, Europe, Life Technologies.

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For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.© 2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. TaqMan is a registered trademark of Roche Molecular Systems, Inc. Printed in the USA. CO21675 0611

Ordering informationProduct Cat. No.

TaqMan® Array Human Viral Pathogen Screening Cards 4465485

extremely high levels of viral nucleic acid not expected to be encountered during standard testing. Comparable assay performance was observed with TaqMan® Array Human Viral Pathogen Screening Cards, which contain all of the assays in a convenient pre-dispensed 384-well format.

In summary, we have documented the establishment and validation of new tools and protocols for the rapid and sensitive detection of viral pathogens in human cell lines for research. These

new TaqMan® Assays help provide a rapid, reliable method for standard monitoring of cell line purity. They are also available loaded into convenient 384-well TaqMan® Array Cards that offer a highly streamlined workflow. These products could be particularly useful tools for researchers to ensure that human pluripotent stem cell cul-tures and their differentiated progeny, particularly from novel sources, are pathogen-free.