# Advancements in Research on Duck Tembusu Virus Infections
## 3 Diagnostic Methods  
Detection methods for DTMUV include virus isolation, multiplex RT-PCR techniques, enzyme-linked immunosorbent assay (ELISA), immunomodtoratographic strip (ICS) assays, and nanoparticle-assisted PCR methods. While virus isolation and identification methods are classic and accurate, their longer turnaround times make them more suitable for experimental research rather than rapid clinical diagnostics. [PERSON] et al. developed a one-step reverse transcription PCR targeting the highly conserved region of DTMUV's NS5 gene, offering high accuracy, specificity, and sensitivity for routine DTMUV detection and epidemiological monitoring. It can widely detect all DTMUV clusters and is capable of detecting DTMUV as low as 0.001 50% embryo lethal dose per milliliter [97]. Given the development of mixed farming systems, increased human and animal mobility, and environmental contamination concerns, duck viral infections have become increasingly serious in recent years, often involving mixed infections. Therefore, establishing a multiplex RT-PCR method is essential for the rapid diagnosis of common duck viral diseases and cost-effective testing. Dual qPCR based on TaqMan enables the rapid diagnosis of DTMUV and Goose Astrovirus Genotype 2 (GoAstV-2), with detection limits of 100 copies/\(\mu\)L and 10 copies/\(\mu\)L, respectively [98]. [PERSON] et al. established a multiplex qPCR capable of simultaneously detecting six duck-origin viruses, including DTMUV. The multiplex qPCR system can amplify six DNA fragments from the combined viral genome when the template concentration is 10\({}^{2}\) copies/\(\mu\)L, and it can specifically detect the nucleic acids of six duck-sensitive viruses, improving efficiency, reagent conservation, and cost savings [99]. [PERSON] et al. developed a multiplex qPCR assay capable of simultaneously detecting Duck Circumvirus (DuCV), DTMUV, Muscovy Duck Reovirus (MDRV), and Novel Duck Reovirus (NDRV). However, it does not amplify other viruses, including Duck Viral Enteritis Virus (DVE), Infectious Bursal Disease Virus (IBDV), Avian Rotavirus (ARV), H5 Avian Influenza Virus (H5 AIV), H7 Avian Influenza Virus (H7 AIV), H9 Avian Influenza Virus (H9 AIV), Newcastle Disease Virus (NDV), and Muscovy Duck Parvovirus (MDPV). The detection limits for DuCV, DTMUV, MDRV, and NDRV are 1.51 \(\times\) 101 copies/\(\mu\)L [100]. The developed multiplex digital PCR (dPCR), compared to multiplex qPCR, can specifically detect DTMUV, DuCV, and NDRV but cannot amplify MDRV, MDPV, Goose Parvovirus (GPV), H4 Avian Influenza Virus (H4 AIV), H6 Avian Influenza Virus (H6 AIV), or NDV. Multiplex dPCR exhibits high sensitivity, with a detection limit as low as 1.3 copies/\(\mu\)L, which is 10 times lower than that of multiplex qPCR [101]. In serological testing, ELISA is commonly used to detect DTMUV antigens and antibodies. An Epitope-ELISA serological diagnostic method was developed using the specific antigenic epitope YAEFI of DTMUV's E protein. Antigens based on YAEFI epitopes exhibit highly specific reactivity with serum samples obtained from ducks infected with DTMUV, without any serological cross-reactivity with West Nile virus (WNV), Dengue virus (DENV), and Japanese encephalitis virus (JEV), and can be applied for clinical diagnosis and antibody level evaluation [102]. The ELISA based on purified NS1 protein demonstrates no cross-reactivity with common duck pathogens such as H5N1 avian influenza, NDV, Duck Hepatitis Virus Type 1 (DHV-1), Duck Reovirus (DRV), Duck Plague Virus (DPV), and Riemerella anantigestifer (Duck Septicemia Agent). This ELISA can be used for the large-scale diagnosis and serological monitoring of DTMUV infection in China [103]. Based on the closed ELISA of monoclonal antibodies (MAbs) against TMUV, the specificity and sensitivity were 96.37% and 100%, respectively, with a kappa value of 0.966. This provides a reliable and rapid diagnostic tool for the serological monitoring and evaluation of TMUV infection [104].