TỐI ƯU HÓA QUY TRÌNH PHÁT HIỆN Hepatopancreatic parvovirus (HPV) VÀ Monodon baculovirus (MBV) TRÊN TÔM BẰNG PHƯƠNG PHÁP DUPLEX REALTIME PCR

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Tác giả: Phạm Phương Thảo Nguyễn, Phạm Trúc Phương Nguyễn

Ngôn ngữ: vie

Ký hiệu phân loại:

Thông tin xuất bản: Tạp chí Khoa học và Công nghệ - Đại học Thái Nguyên, 2023

Mô tả vật lý: tr.299-307

Bộ sưu tập: Metadata

ID: 339682

Thêm vào giỏ Liên kết toàn văn

Both MBV (Monodon baculovirus) and HPV Hepatopancreatic parvovirus) are shrimp enteric viruses that infect intestinal and hepatopancreatic epithelial cells to cause significant mortalities and depressed growth in infected larval, postlarval, and early juvenile stages of shrimp, and thus present a risk to commercial aquaculture. Therefore, in this study, a procedure for detecting of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in shrimp by duplex realtime PCR assay using TaqMan probes in a one-tube PCR reaction has been developed to quickly and accurately detect virus. The optimized duplex realtime PCR protocol include concentrations of primers HPVth/MBV 400 nM/400 nM, concentrations of probe HPV-P1/MBV-P1 là 300 nM/300 nM with the following conditions: initial denaturation at 95°C for 5 min, followed by 40 cycles of amplification at 95°C for 15s and annealing/extension at 60°C for 1 min. The results of evaluating of method show that the LAMP method have sensitivity (SE) is 100%, accuracy is (AC) 100%, specificity (SP) is 100%, the false positive rate is 0% and false negative rate is 0%.MBV (Monodon baculovirus) và HPV (Hepatopancreatic parvovirus) đều là virus ký sinh trên tế bào biểu bì gan tụy và tế bào biểu bì ruột của tôm, gây chết tôm ở giai đoạn ấu trùng Zoea, mysis và tôm giống nhỏ,… nên ảnh hưởng nghiêm trọng đến ngành công nghiệp nuôi tôm. Vì vậy, nghiên cứu này đã phát triển quy trình duplex realtime PCR bằng cách sử dụng đầu dò TaqMan để phát hiện nhanh và chính xác đồng thời monodon baculovirus (MBV) và hepatopancreatic parvovirus (HPV) trong cùng 1 phản ứng. Sau quá trình tối ưu các thành phần và điều kiện cần thiết như tỉ lệ mồi HPVth/MBV 400 nM/400 nM, tỉ lệ probe HPV-P1/MBV-P1 là 300 nM/300 nM với điều kiện phản ứng như sau: 94ºC trong 5 phút, tiếp theo 94ºC trong 15 giây, 60ºC trong 60 giây, chu kỳ này được lặp lại 40 lần. Kết quả nghiên cứu cho thấy phương pháp được xây dựng có độ đặc hiệu (SP) đạt 100%
độ chính xác (AC) đạt 100%
độ nhạy (SE) đạt 100%
tỷ lệ âm tính giả 0% và tỷ lệ dương tính giả 0%, LOD50 là 5 copy/phản ứng.

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