Tilapia lake virus disease (TILVD) can infect all stages of tilapia's growth, with rapid onset and high mortality, and there is currently no specific vaccine. Recently, Zhang Xianpeng's team from Dongguan Animal Disease Prevention and Control Center has researched and established a droplet reverse transcription digital PCR (RT-DDPCR) detection method for tilapia lake virus with high sensitivity, strong specificity and good reproducibility, which is the first report at home and abroad. The minimum number of virus molecules detected by this method is 2 copies, which is more sensitive than other molecular biology detection methods established at home and abroad, and can provide technical support for the quantitative detection of tilapia lake virus and the calibration of standard samples. 01 Tilapia Lake virus droplet reverse transcription digital PCR detection
(1) Establishment of RT-ddPCR detection method
Referring to the whole gene sequence of the 3rd segment of TILv landed in GenBank, the hypothetical protein gene gene gene was selected as the target gene, and 1 pair of primers and probes were designed and synthesized, using the cDNA of TILv as a template, and the annealing temperature was 542, the established TILV RT-DDPCR amplification reaction had the highest amplification efficiency, the most obvious distribution boundary of the negative and negative droplets, and the average copy number was higher. (2) Standard curve constructionThe linear relationship between RT-DDPCR and real-time fluorescence RT-PCR detection method in the range of 1 90 000 copies L was good (Y= 3.).7072x+39.894，r2=0.9958）。(3) Sensitivity, reproducibility, and specificity experimentsThe established RT-DDPCR method has a detection limit as low as 2 copies of L and high sensitivity
The coefficient of variation of the detection was low (486%), with good repeatability;
There was no cross-reactivity with the positive samples of other five common aquatic animal disease viruses (carp edema virus, koi herpes virus, grass carp hemorrhagic virus, crucian carp hematopoietic organ necrosis virus, and cytoswelling iridescent virus), and the specificity was strong.
(4) Clinical sample testingThe test results of 48 clinical samples of tilapia were all negative, and 3 of the 5 proficiency testing samples were positive, which was consistent with the satisfactory results of proficiency testing.
The microdroplet reverse transcription digital PCR method of tilapia lake virus established in this study has high sensitivity, strong specificity and good reproducibility, and it takes about 3 hours from sample pretreatment to the issuance of results, which can be used as a quantitative detection technology for tilapia lake virus. In addition to the accurate and quantitative detection of tilapia lake virus, this detection method can also be applied to the calibration of tilapia lake virus standard samples. It will provide a useful reference for the study of tilapia lake virus.
02 Why did you choose to test for Tilapia Lake virus?
Tilapia lake virus is an RNA virus of the family Orthomyxoviridae, which can infect all stages of tilapia's growth, and has a rapid onset and high mortality, and there is no specific vaccine for it. In 2020, China's Ministry of Agriculture and Rural Affairs and the General Administration of Customs jointly issued Announcement No. 256, listing tilapia lake virus disease (TILVD) as a Class II animal disease in the List of Quarantine Diseases for Imported Animals of the People's Republic of China. At present, electron microscopy observation, virus isolation and culture, and various molecular biology detection methods such as RT-PCR, nested RT-PCR, half-nested RT-PCR, and real-time quantitative RT-PCR have been reported at home and abroad, but in the early stage of infection, the virus content in fish is low and is easy to be ignored, so there is a greater need for detection methods with strong specificity and high sensitivity. 03 About digital PCR detection technology
Concept: Digital PCR technology is a quantitative detection technology developed on the basis of fluorescent PCR detection methods, called third-generation PCR technology. In 1999, Vogelstein and Kinzler first proposed Digital PCR (DPCR), which can convert exponential functions into linear functions, limit dilution of the template, and accurately quantify it with or without fluorescence signal. Digital PCR detection technology is a technological innovation of traditional PCR methods, which can absolutely quantify the copy number of nucleic acids. ApplicationsDigital PCR technology was first applied to the detection of gene mutations in fecal samples of colorectal cancer. In recent years, droplet digital PCR technology has been widely used in the clinical diagnosis of food pathogenic bacteria, animal-derived components, genetic diseases, and infectious diseases. Because it can be quantitative, in the evaluation of the effect of antiviral drugs, the quality and effect of the drug can be intuitively reflected through the comparison of the virus content before and after. The sensitivity of digital PCR is about 100 times higher than that of fluorescent PCR technology, and it can be directly detected without culture in the detection of pathogenic bacteria, which improves the detection rate and reduces the risk of laboratory biosafety. At present, digital PCR detection technology has been applied to the detection of dengue fever, brucella, African swine fever, H5 subtype avian influenza, bovine coronavirus, norovirus, new coronavirus, etc. In the detection of aquatic animal diseases, there are also reports of the application of cyberian herpes virus type 2 and infectious salmon anemia virus.
04 How can droplet digital PCR achieve accurate nucleic acid quantification?
The key is micro-dropping (Figure 1). Droplet digital PCR divides the 20 L reaction into 20 000 uniformly sized droplets, each with a volume of 1 nL. 
Fig.1 The key to accurate nucleic acid quantification by droplet digital PCR—microdroplet note: APreparation of samples;b.Samples are added to the droplet generation card;c.Generated droplets;d.The droplets were simultaneously PCR amplified;e.Record the fluorescence signal of positive and negative droplets in the sample;f.The results of the positive sample are displayed.
All 20,000 droplets were simultaneously PCR amplified in a reaction tube. The droplet analyzer examines the droplets one by one and ensures accurate and repeatable results. The droplet analyzer automatically takes a sample and arranges all the droplets in a single row in the sample, and the droplets pass through the two-color fluorescence detector one by one, and the detector reads the fluorescence signal of each droplet in turn at a speed of 1 000 droplets, and at the same time the droplets are read, Quantasoft software records the fluorescence signal of the positive and negative droplets in each sample and displays them graphically. If the droplet contains a target sequence, the TaqMan probe-labeled fluorescence signal can be detected after amplification. If the droplet does not contain the target sequence, the specific fluorescence signal will not be detected after the PCR reaction is over. By recording and analyzing the fluorescence signal of each droplet, droplet digital PCR enables high-precision nucleic acid quantification with detection sensitivity up to a single copy. After amplification is completed on a common PCR instrument, the amplification of each droplet is tested as an endpoint. Droplets containing the target sequence are positive, and droplets that do not contain the target sequence are negative. The concentration or amount of the target sequence in the sample is determined by the proportion of positive droplets. 
Laboratory research team.
Related Articles:
Min Li, Yongfu Li, Yuhao Huang, Zhuojun Chen, Zuanlan Mo, Qunfang Zhong, Benwang Li, Xianpeng Zhang*Establishment of a digital PCR method for the detection of tilapia lake virus droplet reverse transcription[J].Southern Aquatic Sciences. doi: 10.12131 20220184 Click "Read More" below to learn more.
Written by |Zhang Xianpeng.
Edit |Yan Shuai.
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