Hi Manuchar,

I guess you want to make your own duplex PCR for E. Coli spp and an internal control.

For this you have to find the conserved region of E. Coli spp. And your internal control target must be something different than a bacteria. I used to incorporate Phocide Herpes Virus as internal control.

Furthermore, after developing and testing your primer and probes (incl appropriate fluphore and quenchers), you have to make sure that there are no cross reactivity with any other species and finally validate your duplex-PCR against 400 to 1000 patient sample prospectively/retrospectively.

In order to give you an idea how I have done this in the past, I have included a manuscript of mine on dermatophytes. You could do the same procedures but on E. Coli.

Should you be needing any more assistance and information, do not hesitate to ask again.

Good luck,

Ali

Thank you Ali such a helpful information... i am working in a private company. We are intending to offer customers custom PCR assay. we have two choise one what you have explained and the other is apply  TaqMan PCR assays.  

In the second case i have a problem in selecting right assay.

There are offered great number of assays even for  each pathogene, there are too many strains and purpose  might be detection, genotyping or quantitative. 

Each assay has its Genbank Nomber  by  which I can use to lsearch gene deffinition on  NCBI. 

If you have any experiencein assay serching  please instruct me.

Thank you 

Hi Manuchar,

You are welcome and allow me to make one thing clear. The manuscript that I have sent you is an in-house Taqman PCR assay.

The search for your assay depends on the questions and diagnostics needs of the laboratory. For example the assay you need to choose, must answer the relevant clinical information on how an infection needs to be treated. There are three types of assay’s:

1.      Qualitative: knowing whether a micro-organism is present or not

2.      Quantitative: How much of a micro-organism is present

3.      Genotype: which type of the micro-organism is present

Take sexually transmitted infection (STI) such as Chlamydia in men. First you do a qualitative test/assay (Taqman assay in combination with other micro-organisms such as Neisseria and Trichomonas). If the results are positive for Chlamydia,  you need to do an additional genotyping for LGV-typing. The reason for this is that the LGV-type is clinically treated differently than the other Chlamydia strains.

A quantitative assay you do when you need to know the titration or the viral load for example.

Does this help You? Otherwise we can talk some more.

Good luck,

Ali