Identification of Microorganisms Using RT-PCR

[Makky]

Hi guys, I'll use RT-PCR device to identify some bacterial species on the 16S rRND level using F and R primers. DNA was extracted from the bacterial samples and used as a template and purified DNA containing the same primer sequence will use as a reference for comparison the samples results and the reference result. The primer sheet reported that the Tm (melting temperature) of primer is 60.4C and the PCR protocol run using SYBR Green-1 using melting curve analysis.

I need to know exactly If my sample is amplified and was specific and closely related to the reference, Is the sample must give the same Tm that for primer sheet (60.4 C) from melting curve?? or How I Identify my bacterial samples?? OR record the new one for reference and compare it to the sample??

Plz, I need breifly help in this point.

 

Many thanks,

[CharonY]

OK, melting point analyses won't give you much information as the 16s region is highly conservered and you will not see noticeable shifts between most bacteria.

What you want to measure is the fluorescence curve obtained in from your machine. If the product is amplified you will see an increase in fluorescence. You will then determine the crossing point to determine relative abundance.

 

In order to see what bacteria you got you'll have to sequence the PCR products and build trees from the sequence alignments. Alternatively you can use highly specific primers (but then you might run into trouble if you got bacteria with yet unsequenced rRNA sequences.