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If you have dead listeria can you get a Presumptive Positive?


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#1 HokeyPokey

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Posted 07 February 2018 - 07:48 PM

Question: If you have dead listeria can you get a Presumptive Positive.

 



#2 Charles.C

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Posted 07 February 2018 - 08:42 PM

Question: If you have dead listeria can you get a Presumptive Positive.

 

How do you determine "presumptive positive" ?

 

If the method relies on growth and all the listeria species present have been inactivated then presumably NO.

 

It could also be a false positive perhaps ?


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Charles.C


#3 MWidra

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Posted 07 February 2018 - 09:23 PM

Question: If you have dead listeria can you get a Presumptive Positive.

If your presumptive test is a PCR, then you could get a positive result with all the Listeria dead. A PCR would detect the DNA from the bacteria which would persist even if there is no viable Listeria present. People like it because it is fast, but you would need to verify the result, hence it is a presumptive test.

 

Most ELISA tests are designed to require  a enrichment step where you grow up bacteria to cross the detection threshhold. So, it depends on what method you use.

 

Martha


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#4 FurFarmandFork

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Posted 08 February 2018 - 01:28 AM

If your presumptive test is a PCR, then you could get a positive result with all the Listeria dead. A PCR would detect the DNA from the bacteria which would persist even if there is no viable Listeria present. People like it because it is fast, but you would need to verify the result, hence it is a presumptive test.

 

Most ELISA tests are designed to require  a enrichment step where you grow up bacteria to cross the detection threshhold. So, it depends on what method you use.

 

Martha

Was going to share this. Met with someone from Roka bioscience recently as we were discussing false positives via PCR. Typically PCR requires at least 100 organisms worth of target DNA to indicate a positive. So to their point, if your sample had SO MUCH dead listeria that it still caused a positive, you likely still had a problem but either abused the sample or sanitized a biofilm.


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#5 Charles.C

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Posted 08 February 2018 - 05:50 AM

Was going to share this. Met with someone from Roka bioscience recently as we were discussing false positives via PCR. Typically PCR requires at least 100 organisms worth of target DNA to indicate a positive. So to their point, if your sample had SO MUCH dead listeria that it still caused a positive, you likely still had a problem but either abused the sample or sanitized a biofilm.

 

Hi 3F,

 

Not my area but figures << than "100" have been published (and probably much >> also). eg -

 

Attached File  PCR assay for L.mono in food and environment samples..pdf   2.81MB   21 downloads

 

I also noticed this review which illustrates the wide range of PCR sensitivities -

 

Attached File  Rapid detection methods for bacterial pathogens,2016.pdf   679.94KB   23 downloads

 

 

 

 

 

 

.


Kind Regards,

 

Charles.C


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#6 FurFarmandFork

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Posted 08 February 2018 - 05:53 PM

Hi 3F,

 

Not my area but figures << than "100" have been published (and probably much >> also). eg -

 

attachicon.gif PCR assay for L.mono in food and environment samples..pdf

 

I also noticed this review which illustrates the wide range of PCR sensitivities -

 

attachicon.gif Rapid detection methods for bacterial pathogens,2016.pdf

 

 

 

 

 

 

.

 

My points with the roka people as well. They were also confused when I asked about units. E.g. in the poster you linked, level of detection is listed as a minimum of 10 genomic copies.......but doesn't say per what volume is used....previously I used PCR that had sample wells with 100µl of material...

 

It's complicated and it depends.


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#7 MWidra

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Posted 08 February 2018 - 06:27 PM

My points with the roka people as well. They were also confused when I asked about units. E.g. in the poster you linked, level of detection is listed as a minimum of 10 genomic copies.......but doesn't say per what volume is used....previously I used PCR that had sample wells with 100µl of material...

 

It's complicated and it depends.

PRC sensitivities are typically expressed as the number of copies that can be successfully amplified by the primers and detected by the system used. The units are not concentration, but how many copies it needs in their protocol. It then depends on how the PCR products are going to be measured, and in what instrument, etc. If this is RT PCR, then it will vary based on the label that is used and the sensitivity of the machine.

 

Scientific papers (posters are a publication, in a way) include all the information on how the products were detected, so it is a given that the sensitivity refers to their system and not to any other system.

 

Yes, it's complicated and Yes, it depends. I know, I spent over 30 years in medical research.

 

Martha


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#8 Charles.C

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Posted 09 February 2018 - 04:15 AM

Hi 3F / Martha,

 

Yes, agreed.

 

I saw this comment in another publication for Salmonella/Shigella, half of which i understood -
 

 

When  evaluating  PCR  for  the  detection  of  microorganisms  two  important  criteria  must  be  satisfy:  specificity  and sensitivity [16]. Specificity considered as a way to detect false negatives and sensitivity as to detect false positives. The single PCR using the IS200 or virA primers resulted to be highly specificity and sensitive.

 

The detection level is also an important criterion for evaluating the PCR [17]. Unfortunately, there is no standard for reporting the detection level and some authors indicate it as the grams of DNA or number of cells that can be detected. Due to absence of standard, both criteria were used. In one hand, as little as 10 fg of DNA can be detected. Considering that a typical bacteria carry 6 fg of DNA approximately, this amount of DNA would correspond to two cells. In the other hand, as little as 10 cells can be detected. These values are not far to that published elsewhere [8, 18, 19].

 

 It is well known that different substances, among them food, can inhibit the PCR reaction [20]. For this reason, it was necessary to know the maximum volume of diluent that did not inhibit the PCR reaction. For this purpose, the bacterial strains were inoculated in mayonnaise and subsequently diluted in buffered peptone water. This diluent was used as it is commonly employed to enrich salmonellae and shigellae. The volume and dilution factor that did not inhibit PCR were  5 µl and 1/10, respectively. Altogether, these data served as to calculate detection level of the method: 2000 cells/ml. With this detection level it is obvious that the pathogens must be enriched in mayonnaise before being detected by PCR. Therefore,  the  major  concern  for  the  applicability  of  PCR  for  the  detection  of  salmonellae/shigellae  is  that  needs  an enrichment step. Thus, the method has to be considered a hybrid between molecular and microbiological techniques. Despite of this, detection time with the PCR technique is shortest than the detection time with the traditional technique. This latter needs 3 days to be accomplished while the former would need, according to our estimations, only fourteen hours to be accomplished.

 

 

(There are now several variants of "direct" PCR of course.)


Kind Regards,

 

Charles.C


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#9 moskito

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Posted 11 February 2018 - 02:08 PM

HI,

 

in the past we have had such discussions with detection of salmonella in cocoa/chocolate. First of all the detectibility is very dependent on matrix. With spiking experiments we could not find a detection of a very low number of cells w/o enrichment of 18h+. -> But in any cases after a positive PCR we would do a confirmation by classical methods.

 

Rgds

Moskito






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