Pcr assays for specific detection of enterocytozoon hepatopenaei

A polymerase chain reaction (PCR) assay capable of specifically detecting Enterocytozoon hepatopenaei (EHP) in a nucleic acid sample, the assay comprising:

• specifically amplifying a gene fragment selected from a shrimp ATP-ADP carrier protein gene an EHP spore gene, or both, and optionally an EHP spore wall gene using a polymerase chain reaction (PCR) method, wherein the step of specifically amplifying comprises
• contacting a nucleic acid sample obtained from one or more shrimp, shrimp feed, or shrimp feed ingredient with one or more first primer pairs,
• wherein each of the one or more first primer pairs are configured to specifically amplify one gene fragment from a gene selected from a shrimp ATP-ADP carrier protein gene an EHP spore gene, or both, and optionally an EHP spore wall gene,
• wherein each of the one or more first primer pairs comprises a first oligonucleotide primer and a second oligonucleotide primer, and wherein the each of the one or more first primer pairs are configured to specifically amplify a different gene fragment,
• wherein the first oligonucleotide primer of each of the one or more first primer pairs is a forward direction oligonucleotide primer adapted for PCR amplification of the one gene fragment and specifically binds the gene, and
• wherein the second oligonucleotide primer of each of the one or more first primer pairs is a reverse direction oligonucleotide primer adapted for PCR amplification of the one gene fragment and specifically binds the gene at a different location on the gene then the first oligonucleotide primer,
• optionally contacting the nucleic acid sample with a second primer pair comprising a third oligonucleotide primer and a fourth oligonucleotide primer,
• wherein the third oligonucleotide primer is a forward direction oligonucleotide primer adapted for PCR amplification of a fragment of a control gene,
• wherein the fourth oligonucleotide primer is a reverse direction oligonucleotide primer adapted for PCR amplification of a fragment of the control gene;
• detecting the presence or absence of an amplified double stranded DNA fragment of the shrimp ATP-ADP carrier protein gene, the EHP spore gene, or both, and optionally the EHP spore wall gene in the nucleic acid sample;
• and
• optionally detecting the presence or absence of an amplified double stranded DNA fragment of the control gene in the nucleic acid sample.