Universal primers and the use thereof for the detection and identification of amphibia/fish species
The invention relates to oligonucleotides and to the use thereof as universal primers for the detection and identification of amphibia /fish species, especially in complex and deteriorated substrates. The invention also relates to a method for detecting and identifying amphibia /fish species in samples collected from the environment (ground, water, faeces) or from industry (especially transformed products). The invention further relates to a kit for said detection.
1. A method for amplifying a region of the mitochondrial 12S gene of batrachian and fish species, comprising the following steps:
a) a sample suspected of containing DNA from a batrachian and/or fish species is provided, the batrachian species is a part of the batrachian kingdom of amphibia;
b) an amplification chain reaction is carried out using a pair of oligonucleotides, according to which the first oligonucleotide selectively hybridizes to the sequence SEQ NO. 4 and the second oligonucleotide selectively hybridizes to the sequence SEQ ID NO. 5 or to the sequence SEQ ID NO. 6.
2. A method for detecting a batrachian and/or fish species in a sample, comprising the following steps:
a) a sample suspected of containing DNA from a batrachian and/or fish species is provided, the batrachian species is a part of the batrachian kingdom of amphibia;
b) the total DNA contained in the sample is isolated;
c) an amplification chain reaction is carried out using a pair of oligonucleotides, according to which the first oligonucleotide selectively hybridizes to the sequence SEQ NO. 4 and the second oligonucleotide selectively hybridizes to the sequence SEQ ID NO. 5 or to the sequence SEQ ID NO. 6; and
d) the possible presence of an amplification product is detected.
3. A process for detecting and identifying a batrachian and/or fish species in a sample, comprising the following steps:
a) a sample suspected of containing DNA from a batrachian and/or fish species is provided, the batrachian species is a part of the batrachian kingdom of amphibia;
b) the total DNA contained in the sample is isolated;
c) an amplification chain reaction is carried out using a pair of oligonucleotides, according to which the first oligonucleotide selectively hybridizes to the sequence SEQ NO. 4 and the second oligonucleotide selectively hybridizes to the sequence SEQ ID NO. 5 or to the sequence SEQ ID NO. 6;
d) the presence of an amplification product is detected; and
e) the sequence of the amplification product is determined in order to identify the batrachian and/or fish species contained in the sample.
4. The method of claim 1 , according to which the first oligonucleotide has a nucleotide sequence SEQ ID NO. 1 and the second oligonucleotide has a nucleotide sequence SEQ ID NO. 2 or SEQ ID NO. 3.
5. The method of claim 1 , further comprising the amplification chain reaction carried out with a blocking primer according to SEQ ID NO. 7.
6. The method of claim 2 , according to which the first oligonucleotide has a nucleotide sequence SEQ ID NO. 1 and the second oligonucleotide has a nucleotide sequence SEQ ID NO. 2 or SEQ ID NO. 3.
7. The method of claim 2 , further comprising the amplification chain reaction carried out with a blocking primer according to SEQ ID NO. 7.
8. The method of claim 3 , according to which the first oligonucleotide has a nucleotide sequence SEQ ID NO. 1 and the second oligonucleotide has a nucleotide sequence SEQ ID NO. 2 or SEQ ID NO. 3.
9. The method of claim 3 , further comprising the amplification chain reaction carried out with a blocking primer according to SEQ ID NO. 7.