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  GENIUL

  Advanced PhotoActivation System!

 The latest innovation in Viable PCR for detecting viable bacteria in environmental samples

 

 - PhotoActivation Systemdead organism의 DNA/RNA를 neutralizes 시켜, PCR, Flow Cytometry,

   Fluorescence Microscopy를 이용하여 living cells의 DNA/RNA선택적으로 Detection할 수 있습니다.

 

 - PhotoActivation System allows the activation of a photosensitizer (PS) by low doses of harmless visible

   light of an appropriate wavelength (blue, red, and yellow) to excite the PS to its reactive triplet state,

   which will then generate reactive oxygen species that are toxic to cells.      

 

 

 

Phast BLUE  Assessment of cell viability in fixed cells by Flow Cytometry          

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Phast BLUE  Nonviable cell staining for microscopy detection     

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PAUL Detect only DNA from viable microorganisms    

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Dark Box for sample and reagent incubation during viability PCR process  

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D-Bag Station Decontamination Bag System  For removal of DNA intercalating dyes from aqueous solutions  

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Reaction Buffer for viability PCR , For optimum sample treatment during reagent incubation  

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Reaction Tube Optimal reaction tubes for PhAST Blue platforms

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PEMAX  Reagents, a new step forward in the detection of live cells by PCR

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PEMAX  Monodose TBC-Biomarker Kit        

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Bifidobacterium spp. qPCR detection Kit 

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V-DNA  technology for DNA purification for conventional and viability qPCR   

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 Viability PCR a key tool for early detection of M.tuberculosis in clinical samples

 Viability PCR allow monitoring viable Mycobacteria levels in clinical samples obtaining a more specific and fast diagnostic of disease

 status, but also improving the patients control during treatment. With this approach the treatment success can be detected early

 than conventional methods as culture. The same methodology can be applied for in vitro evaluation of antibiotic resistance, also

 obtaining clear results quick and early than culture approaches.

“Because the detection of viable cells is based on DNA, PMA treatment of clinical samples offers advantages over systems

 that rely on the less stable mRNA or rRNA molecules, with lower sensitivity to poor sample handling.

 The expansion of this work into many other infective contexts is now important, as obtaining more accurate data on

 antibiotic impact could be of significant potential benefit to patients.”

  Roger et al. The exclusion of dead bacterial cells is essential for accurate molecular analysis of clinical samples. Clin Microbiol Infect.

“Here, we demonstrate that the use of PMA treatment to prevent extracellular DNA, or DNA from cells that are nonviable, from

 acting as a template for PCR reactions using total DNA extracted from respiratory samples is both simple and highly effective.”

 Roger et al. Assessing the diagnostic importance of nonviable bacterial cell in respiratory infections. Diagn Microbiol Infect Dis. .

“This PMA-qPCR assay is useful as a rapid 3-day firstand second-line drug susceptibility test for M. tuberculosis.”

 Pholwat et al. Rapid first- and second-line drug susceptibility assay for Mycobacterium tuberculosis Isolates by use of quantitative

 PCR. J Clin Microbiol. 2011 Jan;49(1):69-75.

“PMA real-time PCR is a useful approach for differentiating dead from live bacilli in AFB smearpositive sputum samples.”

 Kim et al. Evaluation of Propidium Monoazide Real-Time PCR for Early Detection of Viable Mycobacterium tuberculosis in Clinical

 Respiratory Specimens. Ann Lab Med 2014;34:203-9

“The assay is useful for monitoring mycobacterial load in pulmonary TB patients during anti-TB treatment.”

  Miotto et al. Early tuberculosis treatment monitoring by Xpert ® MTB/RIF. Eur Respir 2012;39 :1269-71

 

 Viable ( infective ) microorganisms detection by PCR is a practical tool for food microbiology

 Viable real time PCR has been used as an accurate reliable simple method to detect and quantify microorganisms involved in the

 food production process. The technique has been applied in the determination of wine yeasts, Escherichia coli O157:H7,

 Campylobacter jejuni, Zygosaccharomyces bailii, thermophilic Bacillus species, and probiotic bacteria.

 "In conclusion, we now have a new culture-independent methodology for determining microbial diversity in winemaking.”

  Andorrà et al. Determination of viable wine yeast using DNA binding dyes and quantitative PCR. Int J Food Microbiol. 2010 Dec

“ This study, highlights the usefulness of EMA or PMA qPCR as a complement to CFU determination in studying

  bacterial survival after cleaning and disinfection”  

  Marouani-Gadri et al. Potential of Escherichia coli O157:H7 to persist and form viable but nonculturable cells on a food-contact surface

  subjected to cycles of soiling and chemical treatment. Int J Food Microbiol. 2010 Nov 15;144(1):96-103.

“Thus, conventional culturing in conjunction with EMAPCR or viability staining methods may provide a better compromise

  in the detection of viable cells”

  He et al. Quantitative analysis of viable, stressed and dead cells of Campylobacter jejuni strain 81-176. Food Microbiol. 2010

“Thus we feel that we have designed an assay which will be useful for the detection of viable spoilage yeasts in various

 fruit juices.”

 Rawsthorne et al. Detection of viable Zygosaccharomyces bailii in fruit juices using ethidium monoazide bromide and real-time PCR.

“The quantification of spores in the factory milk powders investigated indicates on average the presence of 1.2 log

 units more spores than determined by plate counting”

 Rueckert et al. Rapid differentiation and enumeration of the total, viable vegetative cell and spore content of thermophilic bacilli in milk

 powders with reference to Anoxybacillus flavithermus.

“In conclusion, the PMA real-time PCR and FCM determination of the viability of probiotic bacteria could complement  

 the plate count method which considers only the culturable part of the population."

 Kramer et al. Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry.

 

 A viability-linked metagenomic analysis of cleanroom environments: eukarya, prokaryotes, and viruses                 

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 Viability assay

 Each 500 μl aliquot of filter-concentrated sample suspension to undergo viability assessment was treated with PMA to a final

 concentration of 50 μM [16, 38], mixed thoroughly, and incubated in the dark for 5 min at room temperature.

 Tubes were inverted 5–6 times manually during the incubation to promote homogeneous PMA exposure. Both PMA-treated and

 non-treated samples were subjected to PMA photoactivation at room temperature for 15 min using a LED light source

 (λ = 464–476 nm, 60 W; PhAST Blue, GenIUL, Barcelona, Spain).

 

 Evaluation of viability-qPCR detection system on viable and dead Salmonella serovar Enteritidis

Data Sheet 보기

 One hundred microliters of tested samplewas transferred in a 1.5ml Eppendorf tube. In a dark room (PMA is sensitive to light),

 75 μM or 150 μM of PMA 20 mM was added to each tube. Tubes were incubated in a dark room under rotating agitation for 5

 or 60 min. At the dark incubation step, PMA penetrates the permeable cell-wall/membranes and gets access to the DNA of

 cell-wall/membrane compromised bacteria. Tubes were transferred to the PhAST blue lamp to undergo the LED-light exposure for

 15 min. The light treatment covalently binds PMA to DNA. Free PMA was removed by harvesting the bacteria 10 min at 6000 ×g.

 

 Enhancing the Value of Molecular Methods to the Water Industry: An E. coli Case Study

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 Modifications to Decrease PMA-PCR Method Detection Limit

 The detection limit for the PMA-PCR method was investigated using 90- and 150-CFU E. coli BioBalls and 200- and 250-CFU

 stocks of flow cytometry-sorted E. coli 11775. Because the focus of these experiments was determination of the method

 detection limit, PMA treatment was not applied to these samples. Additional samples with 250 CFU E. coli were heat-killed and

 PMA treated to investigate the effect of PMA at this seed level. The target CFU level of E. coli was added to 100 mL dilute

 PBS (1:100 dilution of 0.1 M PBS) and membrane filtered. The water samples were processed in triplicate using the sample

 processing method outlined in Figure 4.3, with a few modifications. A PhAST Blue photoactivation system (GenIUL) was used

 for 15 minutes to activate PMA in samples

 

 In Vitro and In Vivo Infectious Potential of Coxiella burnetii: A Study on Belgian Livestock Isolates

Data Sheet 보기

 Ethidium Monoazide (EMA)-PCR and Real Time qPCR

 Yolk sacs from C. burnetii-infected embryonated egg were homogenized in PBS buffer before analysis by EMA-PCR.

 Working dilutions were those described in the results section. EMA (Geniul, Spain) was added to a final concentration of 100 mM.

 Samples were then incubated for 30 minutes at 4uC in the dark and vortexed regularly. Subsequently, they were exposed

 to visible light for 30 minutes in an appropriate instrument (PhAST blue system, Geniul).

 

 ‘‘Limits of Control’’ – Crucial Parameters for a Reliable Quantification of Viable Campylobacter by Real-Time PCR

Data Sheet 보기

 EMA/PMA Treatment

 EMA and PMA were kept in stock solutions of 2–10 mM in 20% DMSO at 220uC. Samples of 1 ml were stained with the indicated

 concentration of the dyes. Chicken rinses were either stained directly or centrifuged for 3 min at 80006g and resuspended in 1 ml

 PBS before addition of the dye.  For the establishment of different temperature conditions, the cell suspensions were preincubated

 at the respective temperature for 10 min either in a water bath (25–37uC), in an incubator temperature block (10–20uC) or on ice.

 Subsequently, EMA or PMA was added and the incubation was further prolonged for 5 or 15 min in the dark prior to photo

 activation for 15 min using the PHAST blue system at 100% light intensity (GenIUL). After photoactivation, the samples were

 centrifuged at maximal speed (16.0006g) for 5 min and stored at 220uC, if DNA extraction was not immediately performed.

 

 What does the PhAST Blue or PAUL do?

 The PhotoActivation System for Tubes - PhAST - and PhotoActivation Universal Light - PAUL System - can photo label the

 DNA/RNA of dead organisms through the photoactivation of a mix of photoactivable intercalating dyes with intact and nonintact

 membrane cells by using blue light.

 PhAST Blue system allows simultaneous photoactivation of 12 samples in a simple and efficient manner.

 PAUL system has been designed for working with other sample containers formats as filter membranes, 96 well plates or

 culture bottles.

 What the PhAST Blue or PAUL doesn’t do?

 It is not a detection system by itself. Once the nucleic acid from dead microorganisms has reacted with EMA, PMA and PEMAX

 by photoactivation, a molecular analysis for the target detection needs to be performed e.g. PCR, qPCR, DGGE, pyro-sequencing,

 flow cytometry or fluorescence microscopy.

 Why GenIUL’s systems are the best option for reagents photoactivation?

 Devices based on high power halogen lamps, have been successfully used despite of some serious limitation, e.g. sample

 overheat risk and non-homogeneous light dose.

 Nowadays our systems offer the optimum light spectrum emission for the current vPCR reagents (464 to 476 nm).

 Additionally they allow better control of light dose without sample overheating. Moreover, by the means of its specific

 software it is possible to have a total control of the photoactivation process parameters.

 The use of or systems results in increases the workflow simplicity and reduces experimental variation.

 Who has validated our photoactivation systems?

 At this moment, at lot scientific papers have been published in indexed journals, where our systems were used.

 In our web page (News section), or in our Linkedin group (Viability PCR), you can find links to relevant scientific papers where

 our system was used. 

 Is the PhAST Blue or PAUL light spectrum adequate for PEMAX/ PMA/ EMA photoactivation?

The dominant wavelength of the system is 464-476 nm.

It is important to note that the maximum absorbance for EMA is 460 nm and for the photo-lysed EMA is 475 nm.

In the same way, 470 nm is the optimum wavelength for the PEMAX and PMA photoactivation. So, our systems have a correct

range of wavelength in order to reach good EMA/PEMAX/PMA-nucleic acids cross-linkage.

http://nar.oxfordjournals.org/content/5/12/4891.abstract

What is Viability PCR?

Viability PCR combines the use of photo-reactive reagents with a high affinity for DNA/RNA with a photo-chemical reaction.

The nature of the reagents precludes it to pass through cell membranes. For this reason the DNA from cells with undamaged

membrane will be free of photo blockage. After the treatment of microbial aqueous suspension with our reagents combined

with a photoactivation step, only DNA from live microorganisms will be detected by molecular procedures: PCR, qPCR, DGGE,

Pyro-Sequencing…

How can I prevent the amplification of DNA or RNA from dead microorganisms by using culture-independent methods?

A promising strategy is the use of nucleic acid intercalating dyes which can penetrate membrane damaged cells and are

nearly completely cell membrane-impermeable, such as EMA, PMA and PEMAX in conjunction with molecular detection methods.

What is the scientific concept of the PEMAX Reagent in the viability PCR?

This new reagent, combined with the appropriate reaction buffer, extends the concept of viability PCR to cells with intact

cell membrane structure but also with capability to actively maintain bacterial homeostasis, as a result of active metabolism.

Viability PCR uses cell membrane integrity to differentiate live cells from dead. This new approach improves viability PCR by

enabling it to also discriminate between cells with an intact cell membrane and the ability to actively maintain bacterial

homeostasis and cells that have an intact membrane but are metabolically inactive.

The PEMAX Reagent is a double dye technology patented and developed by GenIUL, S.L. in order to overcome the current

limitations of viability PCR procedures.

Furthermore, the combination of PEMAX Reagent and vPCR buffers is a technology developed by GenIUL.

http://www.sciencedirect.com/science/article/pii/S0890850815000304

 

What does EMA, PMA and PEMAX stands for?

PEMAX is a GenIUL’s trademark.

EMA is the acronym for Ethidium Monoazide

PMA is the acronym for Propidium Monoazide and

What are HighPure - EMA?

This is our brand name for Ethidium Monoazide with a high purity (>95%)

The performance of our reagents isspecifically tested for viable PCR.

What do means “best before” in our reagents?

Most of our reagents are quite stable if are correctly stored, up to now we have experimental data demonstrating their fully reactivity during the specified period. Probably they will be functional after this period however we don’t have evidences to support it.

Can the viability PCR concept be applied as molecular Biomarker?

Yes, it can be applied; viability PCR has potential in monitoring bacterial load in sputum specimens and it has also a role

as a biomarker of cure in TB treatment.

There is an scientific publication about this subject and in which the authors use our technology:

http://www.ncbi.nlm.nih.gov/pubmed/25534168

For this purpose, GenIUL has developed PEMAX Reagent monodoses (TBC-Biomarker kit) (Cat. No. 4900013025)

suitable for use in Mycobacterium tuberculosis biomarker studies.

Can the viability PCR concept be applied for virus detection?

Yes, it can be applied, now by the means of viable PCR we will detect virus with undamaged capsid.

After the treatment of microbial aqueous suspension with our reagents combined with a photoactivation step, only DNA/RNA

from intact viral capsid will be detected by molecular procedures.

 

 

 There exist different scientific publications about this subject. Some of them use our system:

  http://www.ncbi.nlm.nih.gov/pubmed/20599560

  http://www.ncbi.nlm.nih.gov/pubmed/20472736

  http://www.ncbi.nlm.nih.gov/pubmed/21278473

Nucleic acids photo labeling can be used for purpose other than viability PCR ?

Yes, this approach has been successfully used for decades for flow cytometry an fluorescence microscopy.

In our web page you can find specific data sheets.

Is the EMA cytotoxic for live cells?

Although EMA shows higher cytotoxicit than PMA in viable cells, most of the times cytotoxic effects could be reduced or

eliminated with bacterial species dependence, if a right concentration is used. Because EMA shows more efficient penetration

than PMA into membrane-compromised cells, lower EMA concentration should be used.

Moreover, the molecular weight of EMA is lower than PMA molecular weight; therefore if the same w/v concentration is used

for both dyes, there will be more EMA present in the reaction.

Minimum amounts of EMA such as 2.5, 2.3, 1.5, and 1 μg/ml resulted be effective to suppress the DNA amplification from

dead cells when Vibrio vulnificus (Wang and Levin, 2006), Legionella (Chen and Chang 2010), Bifidobacterium (Meng et al. 2010),

and bacterial flora from fish fillet( Lee and Levin, 2009) were analyzed, respectively.

For this reasons the recommended EMA concentration is ≤ 10 mg/mL or ≤ 25 mM.

http://www.ncbi.nlm.nih.gov/pubmed/15932774

http://www.ncbi.nlm.nih.gov/pubmed/20163500

http://www.ncbi.nlm.nih.gov/pubmed/20822571

http://www.ncbi.nlm.nih.gov/pubmed/18817818

On the other hand there exist different papers that demonstrate that EMA is not cytotoxic for yeast.

http://www.ncbi.nlm.nih.gov/pubmed/23132341

http://www.ncbi.nlm.nih.gov/pubmed/21036413

Are the PhAST Blue from GenIUL and the LED Active from IB-AS equivalent systems?

Yes, both systems are equivalent. PhAST Blue from GenIUL is the first evolution and new brand name of LED Active blue

(From IB-applied Science, www.ib-as.com).

PhAST Blue has some user friendliness and software improvements, but internally they share the same hardware design concept.

How does EMA/PMA/PEMAX combined with PhAST Blue or PAUL suppress nucleic acid amplification signal?

A simple sample pre-treatment using EMA, PMA and PEMAX previous to the molecular detection method, is necessary to

only detect DNA from live or infectious microorganism.

The pre-treatment comprises two steps: Step one, mixing of the EMA/PMA/PEMAX reagent with the sample and incubation

in dark conditions, and Step two, sample photoactivation using our PhAST Blue instrument.

The nucleic acid intercalating dyes should only penetrate into membrane compromised cells or dead cells.

The photolysis of EMA, PMA and PEMAX permits cross-linking of the dye to the DNA after exposure to strong visible light,

and in this bound state, the DNA cannot be amplified by molecular procedures.

 http://www.ncbi.nlm.nih.gov/pubmed/15691961

 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1393219/

What is the scientific basis of this technique and our products?

The photo labeling properties of Azide Phenantridium derivatives were published several decades ago. From the 80’s to

today different applications have been published.

 

Flow Cytometry http://www.ncbi.nlm.nih.gov/pubmed/2049970

Viability PCR http://www.ncbi.nlm.nih.gov/pubmed/12703305

Fluorescence microscopy http://www.ncbi.nlm.nih.gov/pubmed/11226693

What is the advisable amount of cells to work with viable PCR?

The optimum maximum amount of cells to work with viable PCR is around 106 cell/ml.

However, an optimization step is always advisable.

 http://www.ncbi.nlm.nih.gov/pubmed/22940102

What is the optimum incubation time?

The incubation of the sample with the reagent on darkness conditions may be critical for some applications.

It is necessary to allow the reagent to entry in all the damaged membrane cells, including spores and protozoan cysts.

For most purposes from 5 to 15 minutes may be satisfactory. In some cases longer incubation times are needed,

for example, 30 minutes for protozoan cyst.

Is the incubation temperature important?

Yes, incubation temperature is important for several reasons:

- Cellular membrane fluidity is strongly influenced by temperature. The active ingredients of our reagents (Specially EMA) are

  quite soluble in hydrophobic solvents, so they have some ability to interact with cell membranes.

  At lower temperature, cell membranes are less fluid, so its specific interaction with the reagent and the subsequent diffusion

  to cytoplasm is greatly reduced.

- Constant temperature incubation minimizes result variability.

- Recent papers suggest that in some cases high temperatures incubation could improve PMA diffusion to dead cells and

  improve the treatment performance.

  http://www.ncbi.nlm.nih.gov/pubmed/23389080

  The Dark Box system is designed in order to protect samples from light and to ensuring constant temperature incubation.

If I work with PhAST Blue, are the tubes material important?

The optimal performance of photochemistry reactions is highly related with theamount of light received by the sample.

For this reason we recommend our reaction tubes (Cat. No. 4900019000) which shows a very a high light transmittance rate.

How can I handle liquid waste with viability dyes ?

Our D-Bag Holder (Cat. No. 900099645) is a safe an easy system for managing these liquid wastes.

If the treatment is not 100% effective, will I have false positive?

A treatment with a 99.9% effectiveness in a sample with 1·105 dead cells may still contain the DNA of 100 dead cells.

Therefore, in some cases, for some techniques with high sensitivity such as nested PCR or real-time PCR, false positive

results can be obtained.

For this reason, some authors recommend a result analysis based on relative rather than absolute values.

http://www.ncbi.nlm.nih.gov/pubmed/20632000

 

As stated below there exist different approaches for optimizing vPCR procedures, one of the more promising is to enhancing

the reaction by the means of specific reaction buffers. In the current GenIUL portfolio you can find reaction buffers for specific

microorganisms.

There exist some practical guidelines for viability PCR procedures optimization?

Yes, you can find a complete review in the next link:  http://www.ncbi.nlm.nih.gov/pubmed/22940102

Does the viability PCR works well with complex samples?

The viability PCR technique has been used with complex samples such as fecal

samples (1) and environmental samples with high turbidity levels (1, 2, 3).

(1) http://aem.asm.org/cgi/content/full/75/9/2940.

(2) http://www.ncbi.nlm.nih.gov/pubmed/19153730

(3) http://www.ncbi.nlm.nih.gov/pubmed/20024544

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