African swine fever (ASF), caused by the African swine fever virus (ASFV), is an acute, hemorrhagic, and contagious disease of domestic pigs and wild boars.  The disease is notifiable and listed by the World Organization for Animal Health (WOAH) (Wang N et al. 2019).  The outcomes of ASF infection can be peracute, acute, subacute, and chronic, depending on the virulence of ASFVs.  According to the report of WOAH (https://www.woah.org/app/uploads/2022/12/asf-report24.pdf), from January 2020 to December 2022, ASF led to more than  2 million pig losses.  Currently, ASFV persists continuously in more than 23 countries and poses a serious threat to the global swine industry.  ASF invaded China on 3 August, 2018, caused by genotype II virulent Georgia-07-like ASFVs (Wen et al. 2019; Zhao et al. 2019; Wang et al. 2020; Wang L et al. 2022).  An experimental study showed that Georgia-07-like ASFV HLJ/18 isolated in China is highly lethal and efficiently transmissible in domestic pigs (Zhao et al. 2019; Jiang et al. 2021).  During the past four years, genotype II Georgia-07-like ASFVs dominantly spread in China.  However, the low virulent genotype II and I ASFVs have been successively reported in China in 2020 and 2021, respectively (Sun et al. 2021a, b; Shi et al. 2022).  Compared with the high virulent genotype II HLJ/18 strain, the low virulent genotype I and II ASFVs had lower virulence and high transmissibility in pigs and induced persistent and chronic infection showing irregular virus shedding at low levels (Sun et al. 2021a, b; Tsegay et al. 2022; Wang P et al. 2022).  Notably, when different genotype I and genotype II viruses infect the same pig in the field, a novel virus may be generated through viral genome recombination, which brings new problems and challenges for the prevention and control of ASF in China.  Thus, a diagnostic method that differentiates genotype I and II ASFVs with high sensitivity and stability is urgently needed and will be helpful for the prevention and control of ASF in China.  

ASFVs have been divided into at least 24 genotypes based on the C-terminus of the B646L gene with 478 nt (Bastos et al. 2003).  B646L gene is one of the most used target genes for ASF diagnosis, which is also the target gene for the WOAH recommended PCR and fluorescent quantitative PCR assays (Agüero et al. 2003; King et al. 2003).  Sanger sequencing of targeted amplification of the B646L genes is the main genotyping approach for ASFVs.  Recently, Li et al. (2022) developed the duplex real-time PCR assay based on the ASFV E296R gene, and Cao et al. (2022) established the TaqMAN-MGB probe assay based on the N-terminal sequences of the B646L gene (Cao et al. 2022; Li et al. 2022), which could distinguish genotype I and II ASFVs with detection limits of 10 copies.  However, the target genes or regions in their methods were out of ASFV genotyping regions.  

Single nucleotide polymorphism (SNP) is a single base change at a specific position in the genome of different individuals and can be used as a genotyping marker for the detection of different individual genotypes (Gut 2001).  The amplification refractory mutation system (ARMS), also named Allele-specific PCR (AS-PCR), relies on the extension of primer only when its 3´ end has a perfect complement to the template (Wang M et al. 2019).  ARMS-qPCR technology has been developed and widely used in SNP detection and genotyping (Ochsenreither et al. 2010; Shi et al. 2013; Wang M et al. 2019).  Compared with other assays for SNP detection and genotyping, ARMS-qPCR has the advantage of low-cost, simple operation, high sensitivity, and rapid and real-time detection.

Here, 126 complete or partial B646L genes of ASFVs, including 78 genotype I and 48 genotype II viruses, were obtained from the GenBank database, and their information is shown in Appendix A.  After analyzing these genes by the MegAlign Software (DNAStar), there were 4 SNPs in the C-terminus of the B646L gene, differentiating genotype I viruses from genotype II viruses (Fig. 1-A).  Two SNPs at sites 1 656 and 1 710 were used to design primers and probes for differential detection of genotype I and II ASFVs (Fig. 1-A).  As previously described (Huang et al. 1992; Liu et al. 2012), primers (I F, II F and R) and probes (probe 1 and probe 2) were designed with the targeted gene sequences using Primer 5 Software (Fig. 1-B; Appendix B).  The duplex ARMS-qPCR reaction system volume was 25 μL: 12.5 μL of 2× HyperProbe Mixture (GENFINE), 0.5 μL of I F, II F and R primers (10 μmol L^–1), 0.5 μL of probe 1 and probe 2 (10 μmol L^–1), 5 μL of template DNA, and 5 μL of ddH₂O.  The duplex ARMS-qPCR was performed by using the Bio-Rad CFX96 Touch Real-Time PCR Detection System with the following reaction conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s.  Fluorescence signal was detected at the end of each cycle of extension step.  For the positive sample of genotype I ASFV, FAM and Cy5 fluorophores could be detected; however, for the positive sample of genotype II ASFV, only FAM fluorophore could be detected (Fig. 1-B).  

The standard curve test revealed that for the standard plasmids of genotype I ASFV, the slopes were –3.3825 for Cy5 and –3.1906 for FAM; the correlation coefficient R² was 0.999 for Cy5 and 0.998 for FAM; the amplification efficiency was 97.53% for Cy5 and 100.06% for FAM, respectively (Fig. 1-C); for the standard plasmids of genotype II ASFV, the slope was –3.2983 for FAM, the correlation coefficient R² was 0.992 for FAM, the amplification efficiency was 100.01% for FAM, whereas Cy5 fluorophore could not be detected (Fig. 1-C).  In addition, the sensitivity of the duplex ARMS-qPCR was 10 copies per reaction for both genotype I and II ASFVs (Fig. 1-D).  Thus, these results indicated that the duplex ARMS-qPCR assay has high efficiency and sensitivity.  

We then evaluated the specificity of the duplex ARMS-qPCR.  The nucleic acids of 7 other swine viruses, including PRRSV, CSFV, PRV, PCV2, PEDV, TGEV, and PoRV, were used as templates.  There were 3 amplification curves obtained for genotype I ASFV (FAM and Cy5 signals) and II ASFV (FAM signal), whereas no amplification curve was recorded for the nucleic acids of PRRSV, CSFV, PRV, PCV2, PEDV, TGEV, and PoRV, as well as genotype II ASFV (Cy5 signal) and ddH₂O (Fig. 1-E).  The results demonstrated that the duplex ARMS-qPCR has a good specificity without cross-reactivity with other swine viruses.

The results of the stable detection limit test showed that for the standard plasmids of genotype I ASFV, all 12 replicates were tested positive at the dilution of 10 copies, while 7/12 replicates were tested positive at the dilution of 5 copies (Fig. 1-F); for the standard plasmids of genotype II ASFV, all 12 replicates were tested positive at the dilution of 10 copies, while 6/12 replicates were tested positive at the dilution of 1 copy (Fig. 1-F).  Thus, the stable detection limit of the duplex ARMS-qPCR was 10 copies per reaction for both genotype I and II ASFVs (Fig. 1-F).

We further assessed the repeatability and reproducibility of the duplex ARMS-qPCR.  The assay tested the standard plasmids of 3 concentrations (10⁶, 10⁴, and 10² copies).  For the standard plasmids of genotype I ASFV, the intra- and inter-assay variation of Ct value for the duplex ARMS-qPCR ranged from 0.07 to 0.93% and 1.2 to 2.17% in FAM fluorescence channel and from 0.38 to 1.02% and 0.85 to 1.27% in Cy5 fluorescence channel, respectively (Table 1).  For the standard plasmids of genotype II ASFV, the intra- and inter-assay variation of Ct value for the duplex ARMS-qPCR ranged from 0.27 to 0.61% and 0.77 to 1.07% in FAM fluorescence channel (Table 1).  These findings suggested that the duplex ARMS-qPCR assay has satisfactory repeatability and reproducibility.

Finally, we evaluated the duplex ARMS-qPCR compared with WOAH-qPCR.  A total of 40 samples were detected using both assays, including blood, oral and rectal swabs, tissues, and cell cultures from pigs or PAMs infected by genotype I and II ASFVs.  Animal studies have evaluated the virulence and transmissibility of genotype I ASFV SD/DY-I/21 and genotype II virus HLJ/18 (Zhao et al. 2019; Sun et al. 2021a), respectively.  The results showed that 36 samples, including 18 of genotype I ASFV and 18 of genotype II ASFV were detected to be positive and differentiated by the duplex ARMS-qPCR, which were consistent with the results of the WOAH-qPCR (Appendix C).  

In summary, we developed a duplex ARMS-qPCR assay based on ASFV genotyping region of B646L gene, which can effectively differentiate genotype I and II ASFVs.  The assay had high sensitivity and specificity and exhibited good results in detecting samples, including blood, oral and rectal swabs, tissues, and cell culture.  Whether our method could be used for differentiating other genotypes of ASFVs is needed for further evalution.  However, just genotype I and II ASFVs are spreading outside Africa.  Thus, our method will provide an additional epidemiological investigation tool to implement effective ASFV control and prevention.

Direct-sowing establishment method has great significance in improving winter oilseed rape (Brassica napus L.) production and guaranteeing edible oil security in China. However, nutrient responses on direct sown winter oilseed rape (DOR) performance and population development dynamic are still not well understood. Therefore, five on-farm experiments were conducted in the reaches of the Yangtze River (RYR) to determine the effects of nitrogen (N), phosphorus (P), and potassium (K) deficiencies on population density, dry matter production, nutrient uptake, seed yield, and yield components of DOR plants. Four fertilization treatments included the balanced NPK application treatment (NPK, 180 kg N, 39.3 kg P, 100 kg K, and 1.8 kg borax ha–1) and three nutrient deficiency treatments based on the NPK treatment, i.e., –N, –P, and –K. The results indicated that DOR population density declined gradually throughout the growing season, especially at over-wintering and pod-development stages. Nutrient deficiency decreased nutrient concentration in DOR plants, limited dry matter production and nutrient uptake, and thereby exacerbated density reduction during plants growth. The poor individual growth and reduced population density together decreased seed yield in the nutrient deficiency treatment. Averaged across all the experiments, seed yield reduced 61% by N deficiency, 38.3% by P deficiency, and 14.4% by K deficiency. The negative effects of nutrient deficiency on DOR performances followed the order of –N>–P>–K, and the effects were various among different nutrient deficiencies. Although N deficiency improved DOR emergence, but it seriously limited dry matter production and nutrient uptake, which in turn led to substantial plants death and therefore resulted in a very low harvested density. The P deficiency significantly reduced initial density, limited plants growth, and exacerbated density reduction. The K deficiency mainly decreased individual growth and yield, but did not affect density dynamic. Our results highlighted the importance of balanced NPK application in DOR production, suggesting that management strategy of these nutrients should be comprehensively considered with an aim to build an appropriate population structure with balanced plant density and individual growth.

Overestimation of nitrogen (N) uptake requirement is one of the driving forces of the overuse of N fertilization and the low efficiency of N use in China. In this study, we collected data from 1 844 site-years of rice (Oryza sativa L.) under various rotation cropping systems across the Yangtze River Valley. Selected treatments included without (N0 treatment) and with N application (N treatment) which were recommended by local technicians, with a wide grain range of 1.5–11.9 t ha–1. Across the 1 844 site-years, over 96% of the sites showed yield increase (relative yield>105%) with N fertilization, and the increase rates decreased from 78.9 to 16.2% within the lowest range <4.0 to the highest >6.5 t ha–1. To produce one ton of grain, the rice absorbed approximately 17.8 kg N in the N0 treatment and 20.4 kg N in the N treatment. The value of partial factor productivity by N (PFPN) reached a range of 35.2–51.4 kg grain kg–1 with N application under the current recommended N rate. Averaged recovery rate of N (REN) was above 36.0% in yields below 6.0 t ha–1 and lower than 31.7% in those above 6.0 t ha–1. Soil properties only affected yield increments within low rice yield levels (<5.5 t ha–1). There is a poor relationship between N application rates and indigenous nitrogen supply (INS). From these observations and considering the local INS, we concluded there was a great potential for improvement in regional grain yield and N efficiency.

To characterize the β-lactam resistance in veterinary clinical isolates of Haemophilus parasuis, 115 isolates were examined for the β-lactam resistance, the possession of β-lactamase, and the presence of β-lactamase genes. The genetic relationship among isolates was evaluated by pulsed-field gel electrophoresis (PFGE). Overall, the commonly detected resistance phenotypes were resistant to ampicillin (26.09%), penicillin (22.61%), amoxicillin (21.74%), cefazolin (14.78%), cefaclor (12.17%), and cefotaxime (6.96%). These strains showed high minimal inhibitory concentration (MICs) to oxacillin. 20.87% strains produced β-lactamase, and 4.35% strains showed extended-spectrum b-lactamase (ESBL) phenotype. Moreover, 19 strains harboured bla genes including TEM-1 (n=5), TEM-116 (n=10), and ROB-1 (n=5). Significantly, one strain possessed both TEM-1 and ROB-1, and displayed resistance to cefotaxime (MIC=8 mg L-1). The epidemiological analysis of PFGE revealed high genetic diversity among bla-positive isolates. This work shows that TEM- and ROB-type β-lactamases are prevalent in H. parasuis isolates in China.