Application of an AlphaLISA method for rapid sensitive detection of African swine fever virus in porcine serum.

Infection with African swine fever virus (ASFV) causes an acute and highly lethal hemorrhagic disease that has been responsible for huge economic losses in China. To exactly detect the antigen of ASFV, we established a rapid, no-wash, one-step sandwich-type immunoassay based on the amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) using two monoclonal antibodies (mAbs) M-5 and M-6 against ASFV p72. ASFV p72 in samples was captured by biotinylated mAb M-5 connected to the donor bead surface via streptavidin and "sandwiched" by mAb M-6 which was coated onto the acceptor bead. Efficacy and sensitivity trials revealed that the AlphaLISA could detect ≥0.78 ng/ml of purified p72 and with a linear range of 0.78-100 ng/ml. The AlphaLISA was specific for ASFV and did not cross-react with other common pathogenic porcine viruses. Compared with RealPCR ASFV DNA test and ASFV antigen detection kit, the sensitivity of the AlphaLISA evaluated in 60 porcine serum samples was 93% and 100%, respectively. The specificity was 100% and 91.7%, respectively. This study presents a good laboratory diagnostic tool for sensitive and efficient detection of ASFV in porcine serum. KEY POINTS: • MAbs M-5 and M-6 recognized various epitopes of ASFV p72. • The established ASFV p72 AlphaLISA showed well specificity, high sensitivity, and satisfied correlation coefficient.

A quantum dot fluorescent microsphere based immunochromatographic strip for detection of brucellosis.

BACKGROUND: Brucellosis is a serious zoonosis disease that frequently causes significant economic loss in animal husbandry and threatens human health. Therefore, we established a rapid, accurate, simple and sensitive fluorescent immunochromatographic strip test (ICST) based on quantum dots (QDs) for detection the antibodies of Brucella infection animals serum. RESULTS: The test strips were successfully prepared by quantum dot fluorescent microspheres (QDFM) as tracers, which were covalently coupled to an outer membrane protein of Brucella OMP22. The outer membrane protein OMP28 and monoclonal antibodies of OMP22 were separately dispensed onto a nitrocellulose membrane as test and quality control lines, respectively. The critical threshold for determining negative or positive through the ratio of the fluorescent signal of the test line and the control line (HT / HC) is 0.0492. The repeatability was excellent with an overall average CV of 8.78%. Under optimum conditions, the limit of detection was 1.05 ng/mL (1:512 dilution). With regard to the detection of brucellosis in 150 clinical samples, the total coincidence rate of ICST and Rose Bengal plate test (RBPT) was 97.3%, the coincidence rate of positive samples was 98.8%, the coincidence rate of negative samples was 95.3%, the sensitivity of RBPT is 1:32, and no cross reaction with the sera of other related diseases was observed. CONCLUSION: In our present study, the QDFM has promising application for on-site screening of brucellosis owing to its high detection speed, high sensitivity, high specificity and low cost.

Characterization and reverse genetic establishment of cattle derived Akabane virus in China.

BACKGROUND: Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. RESULTS: An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. CONCLUSION: We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

Development of polyclonal-antibody-coated immunomagnetic beads for separation and detection of koi herpesvirus in large-volume samples.

To separate and concentrate koi herpesvirus (KHV) from large-volume samples, a separation method based on immunomagnetic beads (IMBs) coated with polyclonal antibody directed against KHV was developed. After treatment with IMBs, viral DNA was extracted from samples and used as a template for quantitative PCR (qPCR). The results showed that the concentration of the template DNA extracted from the virus that had been separated using IMBs was 9.65-fold higher than that from virus not treated with IMBs. The detection limit of the IMBs/qPCR method was found to be at least 10 times lower than that of qPCR alone.

Identification and Molecular Analysis of Ixodid Ticks (Acari: Ixodidae) Infesting Domestic Animals and Tick-Borne Pathogens at the Tarim Basin of Southern Xinjiang, China.

Livestock husbandry is vital to economy of the Tarim Basin, Xinjiang Autonomous Region, China. However, there have been few surveys of the distribution of ixodid ticks (Acari: Ixodidae) and tick-borne pathogens affecting domestic animals at these locations. In this study, 3,916 adult ixodid ticks infesting domestic animals were collected from 23 sampling sites during 2012-2016. Ticks were identified to species based on morphology, and the identification was confirmed based on mitochondrial 16S and 12S rRNA sequences. Ten tick species belonging to 4 genera were identified, including Rhipicephalus turanicus, Hyalomma anatolicum, Rh. bursa, H. asiaticum asiaticum, and Rh. sanguineus. DNA sequences of Rickettsia spp. (spotted fever group) and Anaplasma spp. were detected in these ticks. Phylogenetic analyses revealed possible existence of undescribed Babesia spp. and Borrelia spp. This study illustrates potential threat to domestic animals and humans from tick-borne pathogens.

Development of a droplet digital PCR assay for sensitive detection of porcine circovirus 3.

Porcine circovirus 3 (PCV3), a newly emerged circovirus, is associated with porcine dermatitis and nephropathy syndrome, reproductive failure and multi-systemic inflammation disease, and is widely distributed in pig populations worldwide. Therefore, developing specific diagnostic assays will be important for controlling this emerging pathogen. In this study, we developed a novel droplet digital PCR (ddPCR) assay targeting the PCV3 cap gene to improve the sensitivity of PCV3 detection. The established assay is highly specific to PCV3, and does not cross react with other important swine pathogens. The assay's detection limit was 1.68 ±â€¯0.29 copies of PCV3 DNA per reaction (n = 8), an approximately 10-fold greater sensitivity than that of our previously developed quantitative real-time PCR (qPCR) assay for the same virus. The ddPCR assay results were highly reproducible, with intra- and inter-assay coefficient of variation values of <9.0%. Of the 239 archived pig tissue and serum samples, 42 tested positive for PCV3 by the ddPCR assay. Among the 42 positive samples, 31 tested positive by the qPCR assay. Notably, PCV3 was detected in the serum samples collected from commercially imported healthy boars from the US, France and the UK during 2011-2017. The overall agreement between the two assays was 95.39% (228/239). Furthermore, the linear regression analysis showed that the ddPCR and the qPCR results were significantly correlated with an R2 value of 0.9945. Collectively, these results indicate that the ddPCR assay is a robust diagnostic tool for sensitive detection of PCV3, even in samples with low viral loads.

Comparative evaluation of two commercial ELISA kits for detection of antibodies against Akabane virus in cattle serum.

BACKGROUND: Akabane disease (AD), a barrier to international trade for endemic areas with far economic impact on the countries, is caused by Akabane virus (AKAV). Commercial enzyme-linked immunosorbent assay (ELISA) is a commonly used diagnostic technique for AKAV infection, including the IDEXX and IDVET ELISA kits. However, the comparative evaluation of the IDEXX and IDVET ELISA kits has not been published. The object of this study was to evaluate the test performance of the two commercial ELISA kits in detecting serum anti-AKAV antibodies in cattle. RESULTS: With virus neutralization test (VNT) as the "relative gold standard", the diagnostic sensitivity (DSe) was 80.39% (123/153) and 93.46% (143/153) for the IDEXX and IDVET ELISA kit, when suspect samples were included. The diagnostic specificity (DSp) for the IDEXX and IDVET ELISA kit was 93.48% (502/537) and 82.31% (442/537), respectively. CONCLUSION: Both of the tested ELISA kits could be applied to detect antibodies against AKAV in cattle serum. The IDVET ELISA kit had a higher DSe. The IDEXX ELISA kit possessed the higher DSp. These results have important implications if the kits are used to screen herds or individual cattle in surveillance programs, or at border crossings for import-export inspection and quarantine.

Characterization and application of monoclonal antibodies against channel catfish virus (CCV).

Three monoclonal antibodies (MAbs) (27E4, 17H2, 8B6) against channel catfish virus (CCV) were developed by immunizing Balb/C mice. Using indirect ELISA, these MAbs reacted only with CCV and not with three other fish viruses or nine fish cell lines. During western blotting analysis, MAb 27E4 recognized 170 kDa and 47 kDa proteins, while MAb 17H2 and MAb 8B6 recognized 47 kDa and 56 kDa proteins, respectively. Furthermore, a sandwich ELISA was developed for detection of CCV. The detection limit of the test was 105 TCID50/mL.

Establishment and characterization of a heart-derived cell line from goldfish (Carassius auratus).

The goldfish Carassius auratus, a freshwater fish in the family Cyprinidae, was one of the earliest fish to be domesticated for ornamental purposes. A cell line was established from goldfish heart (GH) tissue to create a biological monitoring tool for viral diseases. The GH cell line was optimally maintained at 25 °C in M199 medium supplemented with 10-20% fetal bovine serum. A chromosomal analysis indicated that the cell line remained diploid, with a mean chromosomal count of 100. In viral inoculation assays, significant cytopathic effects (CPEs) were caused by epizootic hematopoietic necrosis virus (EHNV), Andrias davidianus iridovirus (ADIV), and Bohle iridovirus (BIV) infections in the fish cells and the viral titers (average value) of EHNV, ADIV, and BIV in GH cells reached 105.0, 104.5, and 105.0 TCID50/0.1 mL, respectively, within 7 days. However, no CPE was observed in the cells infected with viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), or grass carp reovirus (GCRV). These results suggest that the GH cell line is a valuable tool for studying viral pathogenesis.

Species discrimination in the subfamily Ostertagiinae of Northern China: assessment of DNA barcode in a taxonomically challenging group.

Gastrointestinal nematodes within the subfamily Ostertagiinae (Teladorsagia, Ostertagia, and Marshallagia et al.) are among the most common infections of domesticated livestock. These parasites are of particular interest, as many of the species within this group are of economic importance worldwide. Traditionally, nematode species designations have been based on morphological criteria. However, this group possesses poorly defined species. There is an urgent need to develop a reliable technique that can distinguish species of Ostertagiinae. DNA barcoding has been proved to be a powerful tool to identify species of birds, mammals, and arthropods, but this technique has not yet been examined for identifying species of Ostertagiinae. In this study, a total of 138 mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) sequences from individuals representing 11 species of Ostertagiinae were acquired by PCR for the first time. The specimens were collected from pastoral area of northern China. Genetic divergence analyses showed that mean interspecific Kimura two-parameter distances of COI (13.61 %) were about four times higher than the mean value of the intraspecific divergence (3.69 %). Then, the performance of the COI to identify species of Ostertagiinae was evaluated by identification success rates using nearest neighbor (NN) and BLASTn. The results indicated that the rates of correct sequence identification for COI were high (>80 %) when using the NN and BLASTn methods. Besides, the deep lineage divergences are detected in Teladorsagia circumcincta. Meanwhile, the analyses also detected no genetic differentiation between some species such as Ostertagia hahurica and Ostertagia buriatica. These results indicate that the traditional status of species within Ostertagiinae should be closely examined based on the molecular data.

Establishment from the snout and kidney of goldfish, Carassius auratus, of two new cell lines and their susceptibility to infectious pancreatic necrosis virus.

Goldfish Carassius auratus are commonly used in scientific research and have a significant economic value in the pet trade. In this study, two cell lines were established from the snout and kidney tissue of goldfish, in order to create a biological monitoring tool for viral diseases. Cell lines were optimally maintained at 25 °C in M199 medium supplemented with 15-20 % fetal bovine serum. Chromosomal analysis indicated that both cell lines remained diploid, with a mean chromosomal count of 100. Results of viral inoculation assays revealed that both cell lines shared similar patterns of viral susceptibility and production to infectious pancreatic necrosis virus (IPNV). The viral titers of IPNV in goldfish snout cell line (GFSe) and goldfish kidney cell line (GFKf) reached 10(6.8) and 10(5.9) TCID50/0.1 mL, respectively, within 7 days. The cytopathic effect could be observed when Cyprinid herpesvirus 2 was inoculated into these cells; however, the CPE disappeared after four passages. No CPE was observed in the cells infected by spring viremia carp virus or grass carp reovirus. These newly established cell lines will be a useful diagnostic tool for viral diseases in this fish species and also for the isolation and study of goldfish viruses in future.

Preparation and characterization of a stable BHK-21 cell line constitutively expressing the Schmallenberg virus nucleocapsid protein.

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that predominantly infects livestock such as cattle, sheep, and goats. Its nucleocapsid (N) protein is an ideal target antigen for SBV diagnosis. In this study, a stable BHK-21 cell line, BHK-21-EGFP-SBV-N, constitutively expressing the SBV N protein was obtained using a lentivector-mediated gene transfer system combined with puromycin selection. To facilitate the purification of recombinant SBV N protein, the coding sequence for a hexa-histidine tag was introduced into the C-terminus of the SBV N gene during construction of the recombinant lentivirus vector pLV-EGFP-SBV-N. The BHK-21-EGFP-SBV-N cell line was demonstrated to spontaneously emit strong enhanced green fluorescent protein (EGFP) signals that exhibited a discrete punctate distribution throughout the cytoplasm. SBV N mRNA and protein expression in this cell line were detected by real-time RT-PCR and western blot, respectively. The expressed recombinant SBV N protein carried an N-terminal EGFP tag, and was successfully purified using Ni-NTA agarose by means of its C-terminal His tag. The purified SBV N protein could be recognized by SBV antisera and an anti-SBV monoclonal antibody (mAb) 2C8 in an indirect enzyme-linked immunosorbent assay and western blot analyses. Indirect immunofluorescence assays further demonstrated that the stable cell line reacts with SBV antisera and mAb 2C8. These results suggest that the generated cell line has the potential to be used in the serological diagnosis of SBV.

Identification of a broadly neutralizing epitope within Gc protein of Akabane virus using newly prepared neutralizing monoclonal antibodies.

Akabane virus (AKAV) is characterized by abortion, stillbirth, premature birth, and congenital deformities in livestock and is widely distributed throughout Australia, Southeast Asia, East Asia, the Middle East, and Africa. Gc protein is the major neutralizing target of AKAV and is often considered as an immunogen to prepare neutralizing antibodies. In this study, we prepared and characterized three monoclonal antibodies (mAbs), 4D1, 4E6, and 4F12, against the Gc protein of AKAV (TJ2016 strain). Western blot (WB) and indirect immunofluorescence assay (IFA) analysis proved that the mAbs can react with both the truncated recombinant AKAV Gc protein and the natural Gc protein produced in the AKAV-infected cells. Further research demonstrated that these mAbs possess neutralizing activity. We next defined a neutralizing epitope 1134SVQSFDGKL1142 by screening a panel of overlapping peptides spanning the truncated Gc protein (aa991∼1232) using the generated neutralizing mAbs. Bioinformatic analysis shows that the neutralizing epitope is highly conserved across different genotypes of AKAV. The newly produced neutralizing mAbs and the identified neutralizing epitope in this study enrich the antigenic epitope information of the AKAV Gc protein and could have potential applications in the development of antigen and antibody detection systems that are specific to AKAV.

Expression and purification of the nucleocapsid protein of Schmallenberg virus, and preparation and characterization of a monoclonal antibody against this protein.

Schmallenberg virus (SBV) is a novel orthobunyavirus that primarily infects ruminants such as cattle, sheep and goats. The nucleocapsid (N) protein of SBV has been shown to be an ideal target antigen for serological detection. To prepare a monoclonal antibody (mAb) against the N protein, the full-length coding sequence of the SBV N gene was cloned into pET-28a-c(+) and pMAL-c5X vectors to generate two recombinant plasmids, which were expressed in Escherichia coli BL21 as histidine (His)-tagged (His-SBV-N) and maltose-binding protein (MBP)-tagged (MBP-SBV-N) fusion proteins, respectively. After affinity purification of His-SBV-N with Ni-NTA agarose and MBP-SBV-N with amylose resin, His-SBV-N was used to immunize BALB/c mice, while MBP-SBV-N was utilized to screen for mAb-secreting hybridomas. Six hybridoma cell lines stably secreting mAbs against N were obtained. Clone 2C8 was selected for further study because of its rapid growth characteristics in vitro and good reactivity with recombinant SBV N proteins in enzyme-linked immunosorbent assays. The epitope recognized by 2C8 is located at amino acids 51-76 of the SBV N protein. Western blot analyses showed that 2C8 reacts with both recombinant SBV N proteins and SBV isolates. It is also cross-reactive with the N proteins of genetically related Shamonda, Douglas and Akabane viruses, but not with the Rift Valley fever virus N protein. The successful preparation of recombinant N proteins and mAbs provides valuable materials that can be used in the serological diagnosis of SBV.

Detection and characterization of Bonamia ostreae in Ostrea edulis imported to China.

The protozoan parasite Bonamia ostreae is a destructive pathogen of flat oysters and has been reported to be widespread in Europe and North America. The biological characteristics of this unicellular parasite are still not fully understood. In this study, 104 Ostrea edulis imported from the USA to the Guangdong province of China for consumption were examined for Bonamia infection. PCR assay, combined with restriction fragment length polymorphism, sequencing and BLAST analysis, showed that B. ostreae DNA could be detected in 1 of the 104 oyster samples. Light microscopy revealed Bonamia-like organisms in the oyster. PCR assay and fluorescent in situ hybridization showed that B. ostreae organisms were present and retained their integrity after 4 wk in culture. Acridine orange-ethidium bromide staining indicated that the B. ostreae were still alive. In conclusion, B. ostreae was present in oysters imported to China. More importantly, the parasite was able to survive for at least 4 wk of in vitro culture at 4°C, which further implied a long-term transmission risk of B. ostreae. Considering the wide culture beds of Crassostrea ariakensis and C. gigas in China, and that C. ariakensis and C. gigas are susceptible hosts or reservoirs of B. ostreae, our study highlights the potential risk of introducing B. ostreae by importing O. edulis from a Bonamia endemic area.

Development of a loop-mediated isothermal amplification method for detection of Perkinsus spp. in mollusks.

Perkinsus is a genus of unicellular protozoan parasite responsible for mass mortality of several commercially valuable mollusks. Surveillance and inspection of its epidemiology in the field calls for convenient and rapid detection methods. Here, a loop-mediated isothermal amplification (LAMP) assay was developed to detect the presence of Perkinsus spp. in mollusks. Specific LAMP primers were designed targeting the conserved internal transcribed spacer 2 (ITS-2) region of the rRNA gene of Perkinsus spp. Using ITS-2 recombinant plasmid as a template, we optimized the LAMP reaction system and conditions and then evaluated the analytical sensitivity and specificity of the assay. The LAMP assay was validated using clam samples collected from coastal areas in eastern China and oysters imported to China and compared with the traditional Ray's fluid thioglycollate culture method (RFTM). Our results showed that the LAMP detection method for Perkinsus was successful. The detection limit was 10 copies of plasmid DNA. Compared to the RFTM assay, the LAMP detection method was more sensitive (56 versus 52 positive out of 60 samples). P. olseni and P. marinus from infected hosts were successfully detected by this method. The LAMP method is rapid, sensitive, and specific for Perkinsus spp. detection, and could be used to screen for perkinsosis both on farms and at ports.

Characterization and Double-Antibody Sandwich ELISA Application of a Monoclonal Antibody Against Akabane Virus Nucleocapsid Protein.

BACKGROUND: Akabane virus (AKAV) is a Culicoides-borne Orthobunyavirus that is teratogenic to the fetus of cattle and small ruminant species. OBJECTIVE: This study aimed to develop an effective diagnostic assay for the detection of AKAV using produced monoclonal antibody (mAb). METHOD: First, the mAb against N protein of AKAV was produced and characterized by Western blot (WB) and indirect immunofluorescence (IFA) assays. Then, the linear epitope of AKAV N protein against the mAb was identified and the mAb was applied to establish a double-antibody sandwich ELISA (DAS-ELISA). RESULTS: One AKAV N-reactive monoclonal mAb was generated and designated as 2D3. WB and IFA assays indicated that 2D3 could react with both recombinant N protein and AKAV isolate TJ2016. The linear epitope recognized by mAb 2D3 was located at amino acids 168-182 of AKAV N protein. The DAS-ELISA established on based mAb 2D3 was able to detect both the purified AKAV N protein (with a detection limit of 6.25 ng/mL) and AKAV-infected cell culture supernatant (with a detection limit of 250 TCID50/mL). CONCLUSIONS: Taken together, we successfully prepared a mAb 2D3 against AKAV N protein and identified its corresponding linear epitope, and then established a DAS-ELISA for the detection of AKAV antigen. HIGHLIGHTS: A produced mAb against AKAV N protein was used to define a linear epitope of AKAV and establish a DAS-ELISA for AKAV antigen detection.

Generation of Stable Cell Lines Expressing Akabane Virus N Protein and Insight into Its Function in Viral Replication.

Akabane virus (AKAV) is a world wide epidemic arbovirus belonging to the Bunyavirales order that predominantly infects livestock and causes severe congenital malformations. The nucleocapsid (N) protein of AKAV possesses multiple important functions in the virus life cycle, and it is an ideal choice for AKAV detection. In this study, we successfully constructed two stable BHK-21 cell lines (C8H2 and F7E5) that constitutively express the AKAV N protein using a lentivirus system combined with puromycin selection. RT-PCR analysis confirmed that the AKAV N gene was integrated into the BHK-21 cell genome and consistently transcribed. Indirect immunofluorescence (IFA) and Western blot (WB) assays proved that both C8H2 and F7E5 cells could react with the AKAV N protein mAb specifically, indicating potential applications in AKAV detection. Furthermore, we analyzed the growth kinetics of AKAV in the C8H2 and F7E5 cell lines and observed temporary inhibition of viral replication at 12, 24 and 36 h postinfection (hpi) compared to BHK-21 cells. Subsequent investigations suggested that the reduced viral replication was linked to the down-regulation of the viral mRNAs (Gc and RdRp). In summary, we have established materials for detecting AKAV and gained new insights into the function of the AKAV N protein.

A Novel, Reverse Transcription, Droplet Digital PCR Assay for the Combined, Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus 2 with Swine Acute Diarrhea Syndrome Coronavirus.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread over the world since its emergence. Although the dominant route of SARS-CoV-2 infection is respiratory, a number of studies revealed infection risk from contaminated surfaces and products, including porcine-derived food and other products. The SARS-CoV-2 outbreak has been severely threatening public health, and disrupting porcine products trade and the pig industry. Swine acute diarrhea syndrome coronavirus (SADS-CoV), which was responsible for large-scale, fatal disease in piglets, emerged in 2017 and has caused enormous economic losses in the pig industry. Currently, reverse transcription real-time PCR (RT-rPCR) is the gold standard method for SARS-CoV-2 diagnosis and is most commonly used for SADS-CoV detection. However, inaccurate detection of the SARS-CoV-2 infection obtained by RT-rPCR is increasingly reported, especially in specimens with low viral load. OBJECTIVE: This study aimed to develop an accurate reverse transcription droplet digital PCR (RT-ddPCR) assay for the detection of SARS-CoV-2 and SADS-CoV simultaneously. METHODS: Two pairs of primers and one double-quenched probe targeting the RNA-dependent RNA polymerase (RDRP) region of the open reading frame 1ab (ORF1ab) gene of SARS-CoV-2 and the corresponding ORF1ab region of SADS-CoV were designed to develop the RT-ddPCR assay. The sensitivity, specificity, repeatability, and reproducibility were tested using complementary RNAs (cRNAs) and clinical specimens. RESULTS: The detection limits of RT-ddPCR were 1.48 ± 0.18 and 1.38 ± 0.17 copies in a 20 µL reaction for SARS-CoV-2 and SADS-CoV cRNAs, respectively (n = 8), showing approximately 4- and 10-fold greater sensitivity than the RT-rPCR assay. This assay also exhibited good specificity, repeatability, and reproducibility. CONCLUSION: The established RT-ddPCR assay was shown to be a highly effective, accurate, and reliable method for the sensitive detection of SARS-CoV-2 and SADS-CoV. HIGHLIGHTS: This RT-ddPCR assay could be used to detect both SARS-CoV-2 and SADS-CoV in a sample with one double-quenched probe, and is also the first reported RT-ddPCR assay for SADS-CoV detection.

Infection prevalence and phylogenetic analysis of Perkinsus olseni in Ruditapes philippinarum from East China.

The prevalence of Perkinsus sp. infection in Manila clam Ruditapes philippinarum was investigated in the coastal areas of east China. Thirteen groups of clams were collected from 5 sites: Dandong and Qingdao Bays (Yellow Sea), Weifang Bay (Bohai Sea), and Ningbo and Fuzhou Bays (East China Sea). The clams were tested for perkinsosis infection using Ray's fluid thioglycollate medium culture assay. Perkinsus sp. was found in samples from all 5 sites from May 2008 to May 2009. Infection prevalence ranged from 43.75 to 95.83%, and was significantly higher in October than in May. The only 3 uninfected groups of clams were collected from Weifang Bay, the site farthest from the ocean. There was no difference in the prevalence of infection among the remaining 4 sites. The conserved internal transcribed spacer regions of the ribosomal RNA gene complex in each of the Perkinsus sp. isolates were amplified by PCR. The resulting amplicons were sequenced and phylogenetically analyzed. All the Perkinsus isolates were identified as Perkinsus olseni.