Detection of amplification products

1. A method for detecting an amplification product of a polynucleotide, comprising:

(a) amplifying a polynucleotide template in a reaction mix that comprises:

(i) a first oligonucleotide for priming an amplification reaction, the first oligonucleotide comprising: i. a primer sequence that is complementary to a site in the template, ii. a tail sequence that is not complementary to the template and that is 5′ of the primer sequence, and iii. a quencher or fluorescent label;

(ii) a second oligonucleotide hybridized to the tail sequence of the first oligonucleotide to form a duplex, the second oligonucleotide having a fluorescent label if the first oligonucleotide has a quencher label, or having a quencher label if the first oligonucleotide has a fluorescent label;

(iii) a third oligonucleotide comprising some or all of the primer sequence contained in the first oligonucleotide and not including a quenching or fluorescent label, wherein the third oligonucleotide is capable of hybridizing with the site in the template and with the second oligonucleotide;

(iv) a strand-displacing polymerase; and

(v) the polynucleotide template,

(b) detecting the amplified product of the polynucleotide.

2. The method according to claim 1 , wherein the polymerases is Bst polymerase, large subunit.

3. The method according to claim 1 , wherein the reaction mix comprises a fourth oligonucleotide comprising a sequence for annealing to a priming site on a second location on the polynucleotide.

4. The method according to claim 1 , wherein the reaction mix comprises a fourth, fifth and sixth oligonucleotide, wherein the fourth oligonucleotide is unlabeled and competes with a dimerized labeled fifth and sixth oligonucleotide.

5. The method according to claim 1 , wherein the first oligonucleotide has a quencher label and the second oligonucleotide has a fluorescent label, or the first oligonucleotide has a fluorescent label and the second oligonucleotide has a quencher label.

6. The method according to claim 1 , in the reaction mixture the ratio of the first oligonucleotide to the third oligonucleotide is in the range of 2:8 to 8:2.

7. The method according to claim 3 , wherein the method comprises releasing the second oligonucleotide by extending the fourth oligonucleotide.

8. The method according to claim 1 , wherein the amount of the first oligonucleotide is X/N where X is the primer concentration for optimal amplification of the product in a single-plex reaction and N is the number of different templates for which primer sets are present in the reaction mixture.

9. The method according to claim 8 , wherein X is in the range of 0.1 μM-2 μM.

10. The method according to claim 1 , wherein the amplifying is done by LAMP, or reverse transcription LAMP.

11. The method according to claim 1 , wherein detecting an amplification product of a polynucleotide further comprises detecting an amplification product of multiple polynucleotides in a multiplex reaction mixture.

12. The method according to claim 11 , wherein the multiplex reaction mixture contains an internal standard polynucleotide.

13. The method according to claim 1 , further comprising determining the size of the amplification product.

14. The method according to claim 1 , wherein the first oligonucleotide are present in the reaction mix at a molar ratio of at least a 1:1 ratio.