Primers, kit and method for detecting ehp pathogens of litopenaeus vannamei

A kit for quantitatively detecting EHP pathogens of Litopenaeus vannamei, which comprises a forward primer having a nucleotide sequence as shown in SEQ ID NO:

• 1, a reverse primer having a nucleotide sequence as shown in SEQ ID NO:
• 2, a forward primer having a nucleotide sequence as shown in SEQ ID NO:
• 4, a reverse primer having a nucleotide sequence as shown in SEQ ID NO:
• 5, a probe having a nucleotide sequence as shown in SEQ ID NO:
• 3, and a probe having a nucleotide sequence as shown in SEQ ID NO:
• 6.

A method for quantitatively detecting EHP pathogens of Litopenaeus vannamei, comprising:

• taking the extracted tissue DNA of Litopenaeus vannamei as a sample to be detected;
• and
• placing a PCR reaction system into a PCR reaction tube, placing the PCR reaction tube into a quantitative fluorescence PCR instrument, setting a reaction time, and carrying out a duplex PCR reaction;
• wherein the PCR reaction system comprises a forward primer having a nucleotide sequence as shown in SEQ ID NO:
• 1, a reverse primer having a nucleotide sequence as shown in SEQ ID NO:
• 2, a forward primer having a nucleotide sequence as shown in SEQ ID NO:
• 4, a reverse primer having a nucleotide sequence as shown in SEQ ID NO:
• 5, a probe having a nucleotide sequence as shown in SEQ ID NO:
• 3, a probe having a nucleotide sequence as shown in SEQ ID NO:
• 6, Premix Ex Taq (Probe qPCR) (2×), ROX Reference Dye (50×), and a sample;
• the sample is a sample to be detected, a standard, a negative control or a blank control;
• the standard comprises plasmids containing an 18S rRNA gene of Litopenaeus vannamei at a known concentration and plasmids containing a EHP SWP gene at a known concentration, and meanwhile the standard is used as a positive control;
• the negative control comprises DNAs extracted from tissues of healthy Litopenaeus vannamei;
• and the blank control comprises H2O;
• wherein the 18S rRNA gene of Litopenaeus vannamei is used as an internal reference;
• when both the positive control and the internal reference have amplification product, the blank control has no amplification product, and the negative control only has amplification of the 18S rRNA gene product of Litopenaeus vannamei and has no amplification of the EHP SWP gene product, it indicates that the PCR reaction is normal;
• if the sample to be detected has amplification of the EHP SWP gene product, it indicates that the detection result is positive;
• and
• if the sample to be detected has no amplification of the EHP SWP gene product, it indicates that the detection result is negative;
• and
• linear regression is conducted according to a standard curve and Ct values corresponding to the product to obtain the EHP pathogen content in the sample to be detected.