Detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Litopenaeus vannamei by ramification amplification assay.
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a single-stranded DNA virus that causes developmental and growth abnormalities in Pacific white shrimp Litopenaeus vannamei (also known as Penaeus vannamei). Nucleic acid based methods such as in situ hybridization (ISH) and PCR have been commonly used for IHHNV detection. Ramification amplification (RAM), an isothermal nucleic acid amplification approach, was used in this study to detect IHHNV in L. vannamei. RAM offers many advantages over PCR, including simple procedures and short detection time, and is labor-saving and cost-effective. RAM exponentially amplifies a circular oligonucleotide amplicon (C probe) after a target-specific ligation step through sequential primer extension and strand displacement processes. The conditions of an IHHNV RAM assay were optimized using artificial templates and targets prior to application. Using DNA of IHHNV-infected L. vannamei as targets, results revealed that RAM amplified target DNA with similar sensitivity as PCR. RAM offers competitive levels of speed, simplicity and sensitivity among various pathogen diagnostic methods.
Teng
Ping-Hua
PH
Department of Animal Science and Biotechnology, Tunghai University, Taichung 40704, Taiwan, ROC.
Lee
Pei-Yu
PY
Lee
Fu-Chun
FC
Chien
Hung-Wen
HW
Chen
Man-Shu
MS
Sung
Ping-Feng
PF
Su
Chen
C
Ou
Bor-Rung
BR
D000818:Animals; D004279:DNA, Viral / Q000737:chemistry / Q000302:isolation & purification*; D019223:Densovirinae / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry; D033561:Penaeidae / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D009841:Oligonucleotides