Method of detecting one or more limited copy targets
A method allowing simultaneous amplification of multiple low-abundance targets in environmental samples. This is a two-step process that includes a combined reverse transcription and pre-amplification step, which utilizes a mix of gene-specific primer sets, followed by a second amplification step performed on the previously generated “RT-amplification” product. Initial amplification of each target is performed prior to the splitting of the sample for individual amplification and identification. The method combines the process of reverse transcription and amplification within a single processing apparatus. The method also enables gene-specific reverse transcription using gene-specific primers, thereby reducing if not eliminating non-specific product in this reverse transcription step of the process.
1 . A method of detecting multiple limited copy targets, the method comprising:
a. providing a sample including a plurality of differently sequenced RNA strands;
b. adding multiple different first pairs of gene-specific primers to the sample, wherein each first pair of gene-specific primers corresponds to a specific gene sequence;
c. adding reagents to the sample;
d. incubating the sample at a first temperature for a first period of time such that if one or more of the specific gene sequences are present within the sample, a corresponding gene-specific primer adheres to each specific RNA strand that includes one of the present specific gene sequences, thereby reverse transcribing each specific RNA strand to a corresponding cDNA strand including the specific gene sequence;
e. performing a first thermal cycling process to amplify each specific gene sequence within the cDNA strand according to a linear phase amplification;
f. dividing the sample into portions;
g. adding reagents and one or more different second pairs of gene-specific primers to each sample portion, wherein each second pair of gene-specific primers corresponds to one of the specific gene sequences;
h. performing a second thermal cycling process on each sample portion to amplify the one or more specific gene sequences in each sample portion; and
i. detecting the presence of the one or more specific gene sequences in each sample portion.
2 . The method of claim 1 wherein N different pairs of gene-specific primers are added to the sample such that the presence of up to N specific gene sequences are detected.
3 . The method of claim 2 wherein the sample is divided into N portions and one second pair of gene-specific primers is added to each sample portion.
4 . The method of claim 1 wherein a minimum quantity of the sample provided is greater than or equal to about 2 . 5 femtograms.
5 . The method of claim 1 wherein the second thermal cycling process is performed to saturation.
6 . The method of claim 1 wherein performing the first thermal cycling process is automatically performed after the first period of time.
7 . The method of claim 1 wherein incubating the sample and performing the first thermal cycle are performed within a single containment vessel.
8 . The method of claim 1 wherein the first thermal cycling process and the second thermal cycling process each comprise a polymerase chain reaction.
9 . The method of claim 1 wherein the first thermal cycling process includes five to fifteen cycles.
10 . The method of claim 1 wherein the second thermal cycling process includes at least a number of thermal cycles sufficient for entering into an exponential phase of amplification.
11 . A method of detecting a limited copy target, the method comprising:
a. providing a sample including one or more differently sequenced RNA strands;
b. adding a first pair of gene-specific primers to the sample, wherein the first pair of gene-specific primers corresponds to a specific gene sequence;
c. adding reagents to the sample;
d. incubating the sample at a first temperature for a first period of time such that if the specific gene sequence is present within the sample, one of the first pair of gene-specific primers adheres to a specific RNA strand that includes the specific gene sequence, thereby reverse transcribing the specific RNA strand to a corresponding cDNA strand including the specific gene sequence;
e. performing a first thermal cycling process to amplify the specific gene sequence within the cDNA strand according to a linear phase amplification;
f. adding reagents and a second pair of gene-specific primers to the sample, wherein the second pair of gene-specific primers correspond to the specific gene sequence;
g. performing a second thermal cycling process to amplify the specific gene sequence; and
h. detecting the presence of the specific gene sequence.
12 . The method of claim 11 wherein performing the first thermal cycling process is automatically performed after the first period of time.
13 . The method of claim 11 wherein incubating the sample and performing the first thermal cycle are performed within a single containment vessel.
14 . The method of claim 11 wherein the first thermal cycling process and the second thermal cycling process each comprise a polymerase chain reaction.
15 . The method of claim 11 wherein the first thermal cycling process includes five to fifteen cycles.
16 . The method of claim 11 wherein the second thermal cycling process includes at least a number of thermal cycles sufficient for entering into an exponential phase of amplification.
17 . The method of claim 11 further comprising diluting the sample with a neutral solution prior to performing the second thermal cycling process.
18 . The method of claim 11 wherein the second thermal cycling process is performed to saturation