The establishment of a recombinase polymerase amplification technique for the detection of mouse poxvirus.

BACKGROUND: Ectromelia virus (ECTV) is the causative agent of mousepox in mice. In the past century, ECTV was a serious threat to laboratory mouse colonies worldwide. Recombinase polymerase amplification (RPA), which is widely used in virus detection, is an isothermal amplification method. RESULTS: In this study, a probe-based RPA detection method was established for rapid and sensitive detection of ECTV.Primers were designed for the highly conserved region of the crmD gene, the main core protein of recessive poxvirus, and standard plasmids were constructed. The lowest detection limit of the ECTV RT- RPA assay was 100 copies of DNA mol-ecules per reaction. In addition, the method showed high specificity and did not cross-react with other common mouse viruses.Therefore, the practicability of the RPA method in the field was confirmed by the detection of 135 clinical samples. The real-time RPA assay was very similar to the ECTV real-time PCR assay, with 100% agreement. CONCLUSIONS: In conclusion, this RPA assay offers a novel alternative for the simple, sensitive, and specific identification of ECTV, especially in low-resource settings.

Combined Metabolite Analysis and Network Pharmacology to Elucidate the Mechanisms of Therapeutic Effect of Melastoma dodecandrum Ellagitannins on Abnormal Uterine Bleeding.

The abnormal uterine bleeding (AUB) is complex and usually leads to severe anemia. Melastomadodecandrum (MD) is clinically used for the treatment of metrorrhagia bleeding. The MD ellagitannins (MD-ETs) had been evidenced being effective at hemorrhage, and exerts biological activities upon their metabolites including ellagic acid and urolithins. In this study, the blood-permeated metabolites from theMD-ETs were analyzed using LC-MS approach, and 19 metabolites including ellagic acid and urolithin A derivatives were identified. Furthermore, a network pharmacology analysis including the target prediction analysis, AUB target analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted to reveal the relationships between "metabolites-targets-pathways", which was further verified by molecular docking analysis. The results showed that methyl ellagic acid, urolithin A and isourolithin A produced from MD-ETs can be absorbed into the blood, and might act on the core targets of VEGFA, SRC, MTOR, EGFR and CCND1. And the hemostatic effects were exerted through PI3K-Akt, endocrine resistance and Rap 1 signaling pathways. These results implied the potential effective constituents and action mechanism of MD-ETs in the therapy of AUB, which will promote the application of MD-ETs as natural agent for the treatment of gynecological bleeding diseases.

Molecular networking-assisted systematical profiling and the in vivo neuroprotective effect of ellagitannins from the Melastoma dodecandrum Lour.

BACKGROUND: Ellagitannins (ETs) are a major classification of natural tannins, with relatively large and complex structures. ETs from medicinal plants are focused increasingly due to urolithins, a kind of intestinal metabolite of ETs, which showed promising anti-Alzheimer's disease (AD) effects. Melastoma dodecandrum (MD), a widely used traditional Chinese medicine is rich in ETs, but their chemistry and potential neuroprotective effects have not been investigated. PURPOSE: This study aimed to identify the chemical composition of ETs in the crude extract of MD and to investigate their neuroprotective effects in vivo. METHODS: UPLC-QTOF-MS-based molecular networking (MN) and structural characterization were applied to targeted profiling of the MD-ETs. Animal behavior experiments, including the novel object recognition test (NOR), open field test (OFT), and Morris water maze test (MWM), were conducted to assess the memory improvement effects of MD-ETs in AD model mice. RESULTS: A total of 70 ETs, ranging from monomers to tetramers, were tracked and characterized in the MD extract using MN-guided targeted profiling, with 59 of them reported for the first time in this species. MD-ETs significantly improved memory impairment in AD mice, as indicated by decreased escape latency, increased number of crossings and target quadrant distance in MWM, increased rearing number in OFT, and increased preference index in NOR. CONCLUSION: This study systematically characterized the composition and structural features of ETs in MD using targeted LC-MS profiling, expanding the chemical information of ETs in MD. Furthermore, the results demonstrate that MD-ETs have significant effects on improving impaired memory in AD mice, suggesting their potential as alternative natural medicines for the treatment of neurodegenerative diseases.

Identificationof a novel linear epitope on the porcine reproductive and respiratory syndrome virus nucleocapsid protein, as recognized by a specific monoclonal antibody.

Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) remains one of the most threatening pathogens of swine. The nucleocapsid (N) protein is the major structural protein of the virus and has been used as a PRRSV diagnostic antigen due to its high level of inherent immunogenicity. Methods: The recombinant PRRSV N protein was generated by the prokaryotic expressing system and used to immunized mice. Monoclonal antibodies against PRRSV were produced and validated by western blot analysis and indirect immunofluorescence analysis. In this study, the linear epitope of a specific monoclonal antibody mAb (N06) was subsequently identified by enzyme-linked immunosorbent assays (ELISA) using the synthesized overlapping peptides as antigens. Results: According to the results of western blot analysis and indirect immunofluorescence analysis, mAb (N06) was capable of recognizing the native form as well as the denatured form of PRRSV N protein. The results of ELISA showed that mAb N06 recognized the epitope NRKKNPEKPHFPLATE, which was consistent with BCPREDS predictions of antigenicity. Conclusion: All the data suggested that the mAb (N06) can be used as diagnostic reagents for PRRSV detection, while the recognized linear epitope can be useful in epitope-based vaccines development, which is helpful for the control of local PRRSV infections in swine.

Preparation of a new monoclonal antibody against nucleocapsid protein of swine acute diarrhea syndrome coronavirus and identification of its linear antigenic epitope.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), which causes severe diarrhea in newborn piglets, was first identified in Southern China in 2017. Since the Nucleocapsid (N) protein in SADS-CoV is highly conserved and plays a key role in virus replication, it is often used as a target protein in scientific research. In this study, the N protein of SADS-CoV was successfully expressed, and a new monoclonal antibody (mAb), 5G12, against the protein was generated successfully. The mAb 5G12 can be used to detect SADS-CoV strains by indirect immunofluorescence assay (IFA) and western blotting. The mAb 5G12 epitope was located to amino acids 11 EQAESRGRK 19 by evaluating the antibody for reactivity with a series of truncated N protein segments. The biological information analysis showed that the antigenic epitope had a high antigenic index and conservation. This study will help further understand the protein structure and function of SADS-CoV and in the establishment of specific SADS-CoV detection methods.

Simultaneous Detection of Three Subgroups of Avian Leukosis Virus Using the Nanoparticle-Assisted PCR Assay.

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid detection of pathogens. A sensitive and specific multiple nanoPCR assay was developed for simultaneous detection of avian leucosis virus (ALV) subgroups A, B and J. In this study, three pairs of primers were designed, based on the conserved region of the gp85 gene. An exploration of the optimal primer concentration and annealing temperature were carried out, for better performance of the nanoPCR assay. According to the results, the multiple nanoPCR assay amplified 336 pb, 625 bp and 167 bp fragments of ALV-A, -B and -J, respectively, and showed no cross-reactivity with irrelevant pathogens, suggesting the excellent specificity of the assay. The constructed standard DNA templates were used to estimate the limit of detection. As shown by the results, the detection limit of the nanoPCR assay was nearly 10 copies/µL. To further evaluate the detection ability of the assay, 186 clinical samples were detected using the nanoPCR assay, among which, 14 samples were confirmed as ALV positive; the results were further confirmed by sequencing. In conclusion, a highly specific and sensitive nanoPCR assay was successfully developed, which could be a useful tool for clinical diagnosis as well as for the discrimination of ALV-A, -B and -J.

Single-dose rAAV5-based vaccine provides long-term protective immunity against SARS-CoV-2 and its variants.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus and its variants, has posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against SARS-CoV-2 variants. Therefore, novel vaccines to match mutated viral lineages by providing long-term protective immunity are urgently needed. We designed a recombinant adeno-associated virus 5 (rAAV5)-based vaccine (rAAV-COVID-19) by using the SARS-CoV-2 spike protein receptor binding domain (RBD-plus) sequence with both single-stranded (ssAAV5) and self-complementary (scAAV5) delivery vectors and found that it provides excellent protection from SARS-CoV-2 infection. A single-dose vaccination in mice induced a robust immune response; induced neutralizing antibody (NA) titers were maintained at a peak level of over 1:1024 more than a year post-injection and were accompanied by functional T-cell responses. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines produced high levels of serum NAs against the circulating SARS-CoV-2 variants, including Alpha, Beta, Gamma and Delta. A SARS-CoV-2 virus challenge showed that the ssAAV5-RBD-plus vaccine protected both young and old mice from SARS-CoV-2 infection in the upper and lower respiratory tracts. Whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genomes of vaccinated mice one year after vaccination, demonstrating vaccine safety. These results suggest that the rAAV5-based vaccine is safe and effective against SARS-CoV-2 and several variants as it provides long-term protective immunity. This novel vaccine has a significant potential for development into a human prophylactic vaccination to help end the global pandemic.

Establishment of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of swine acute diarrhea syndrome coronavirus (SADS-CoV).

BACKGROUND: Swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute vomiting and diarrhea in piglets, leading to significant financial losses for the pig industry. Recombinase polymerase amplification (RPA) is a rapid nucleic acid amplification technology used under constant temperature conditions. The study established a real-time reverse transcription (RT)-RPA assay for early diagnosis of SADS-CoV.  RESULTS: The detection limit of the real-time RT-RPA was 74 copies/µL of SADS-CoV genomic standard recombinant plasmid in 95% of cases. The assay was performed in less than 30 min and no cross-reactions were observed with eight other common viruses that affect swine, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudo rabies virus (PRV), swine influenza virus (SIV), seneca valley virus (SVA), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV). The coefficient of variation (C.V.) values of the two standards dilutions and three positive clinical sample ranged from 2.95% to 4.71%. A total of 72 clinical fecal samples from swine with diarrheal symptoms were analyzed with the developed RT-RPA and quantitative RT-PCR. There was 98.61% agreement between the RT-RPA and the quantitative real-time PCR results. CONCLUSIONS: These results indicated that the developed RT-RPA assay had good specificity, sensitivity, stability and repeatability. The study successfully established a broadly reactive RT-RPA assay for SADS-CoV detection.

LC-MS-based identification and antioxidant evaluation of small molecules from the cinnamon oil extraction waste.

Cinnamon oil is obtained by steam distillation from cinnamon leaves and is usually considered highly cost-effective compared to bark oil, however, which results in tons of waste cinnamon leaves (WCL) discarded annually. By using MS/MS molecular networking (MN) assisted profiling, six main chemical diversities including flavonols and flavones, phenolic acids, lactones, terpenoids, phenylpropanoids and flavanols were rapid revealed from WCL aqueous extract. 101 compounds were tentatively identified by assigning their MS/MS fragments within typical pathways under ESI-MS/MS dissociation. The featured phenolic acids, terpenoids and their glycosides in cinnamon species were recognized as the main constituents of WCL. The hydrophilic lactones, lignans and flavanols were reported for the first time in cinnamon leaves. Furthermore, ABTS and FRAP assays integrated with MN analysis were conducted to uncover an antioxidant fraction, from which 40 potential antioxidant compounds were rapidly annotated. This fundamental information will help expand the utilization of WCL from cinnamon oil industry.

Therapeutic effect of continuous diffusion of oxygen therapy combined with traditional moist wound dressing therapy in the treatment of diabetic foot ulcers.

AIMS: To investigate the therapeutic effect of continuous diffusion of oxygen (CDO) combined with traditional moist wound dressing (MWD) on the diabetic foot ulcers (DFUs) inpatients. METHODS: The inpatients from May 2016 to April 2018 were enrolled and randomly divided into MWD, CDO and combination treatment groups (n = 40 each group). The moist dressing was used in the MWD group while a micro-oxygen supply device was used in the CDO group. The combination treatment group was given both MWD and CDO. All patients were treated for 8 weeks. The wound healing, amputation rate, and inflammatory control were evaluated. RESULTS: Compared with MWD and CDO groups, the combination group showed a higher wound healing rate (P < 0.05), lower white blood cell count (P < 0.05) and lower high-sensitivity C-reactive protein level (P < 0.05). During 1-year follow-up, the amputation rate was 0% in combination group, which was significantly lower than that in other two groups (P < 0.05). CONCLUSIONS: The combination of MWD and CDO was effective in promoting healing and preventing infection of DFUs, which holds a potential to be a new strategy for the treatment of this critical clinical condition.

Rapid detection of porcine deltacoronavirus and porcine epidemic diarrhea virus using the duplex recombinase polymerase amplification method.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) have emerged and spread throughout the porcine industry in many countries and are economically important pathogens causing diarrhea in sows and acute death in newborn piglets. Therefore, a sensitive diagnostic method would be beneficial for the prevention and control of PEDV and PDCoV infection. However, traditional detection methods have a number of drawbacks. This research aimed to establish a rapid detection method of duplex recombinant enzyme-mediated thermostatic amplification (RT-RPA) for PEDV and PDCoV. In this study, eight pairs of primers were designed for each virus according to the conserved domains of both PEDV and PDCoV from the NCBI Genbank, and one pair of primers was selected for each virus following the test results. After optimization of the reaction time, reaction temperature and primer concentration ratio, the duplex RT-RPA assay amplified a 226-bp fragment specifically for PEDV and a 321-bp fragment specifically for PDCoV. Meanwhile, the specificity and sensitivity of the primers and clinical samples were tested to verify the establishment of the RT-RPA method. The sensitivities of the duplex RT-RPA method for PEDV and PDCoV were 1 × 102 copies/µL. The results were consistent with PCR results and showed that a detection method for PEDV and PDCoV duplex RT-RPA was successfully established. In summary, the duplex recombinase polymerase amplification method could offer a promising alternative to the duplex RT-qPCR for detection of PEDV and PDCoV.

A review of the traditional uses, phytochemistry and biological activities of the Melastoma genus.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Melastoma consists of approximately 100 species distributed widely in tropical and subtropical countries, and Melastoma species are often used for medicinal purposes, such as treatment for bleeding, diarrhea, diabetes, and gynecological tumors by local people, mostly in Southeast Asian countries. AIM OF THE REVIEW: The present review summarizes the traditional uses, phytochemistry and pharmacology of species belonging to Melastoma to suggest further research strategies and to facilitate the exploitation of the therapeutic potential of Melastoma species for the treatment of human disorders. MATERIALS AND METHODS: Information related to the traditional uses, phytochemistry and pharmacological activities was systematically collected by searching for the word "Melastoma" in electronic databases, including SciFinder, Web of Science, PubMed, and Google Scholar, from Apr. 1968 until Dec. 2019. RESULTS: A systematic literature survey revealed that Melastoma spp. are widely distributed in southern Asia to northern Oceania and the Pacific Islands and are traditionally used to treat bleeding, diarrhea, swelling, and gynecological tumors. Approximately 142 compounds, including flavonoids, tannins, phenylpropanoids, organic acids, terpenoids, and steroids, have been reported from Melastoma spp. Different extracts have been evaluated for their pharmacological activities, including anti-inflammatory, hemostatic, anticoagulant, cytotoxic, antibacterial, antioxidant, hepatoprotective, gastroprotective and hypoglycemic activities. CONCLUSIONS: Melastoma spp. are popularly used in Southeast Asian countries as effective herbs and are rich in flavonoids, tannins and organic acids with valuable medicinal properties. However, additional studies of the chemical constituents and the mechanism-based pharmacological activities of many members of Melastoma are still needed for developing new plant-derived drugs. In addition, studies on the clinical safety and efficacy of Melastoma are also needed.

Application of recombinase polymerase amplification method for rapid detection of infectious laryngotracheitis virus.

Infectious laryngotracheitis is a significant respiratory disease of chickens that causes huge economic losses due to high morbidity and mortality and reduced egg production. A real-time recombinase polymerase amplification (RPA) assay was developed to accurately detect ILTV. The specific probe and primer sets were carefully designed and screened. The real-time RPA assay was carried out at 39 °C for 30 min, and results were obtained within 15 min. The results of the specificity assay showed no fluorescence signals with other avian-related viruses. The sensitivity of the assay was 1 × 102 copies/µL. The low CV value showed that the assay was reproducible. A total of 115 clinical samples were tested using the real-time RPA assay and the real-time PCR assay in parallel; the coincidence rates of the two detection methods were 100%. The results indicated that the real-time RPA assay is a specific, sensitive, rapid, and useful tool for epidemiological studies and clinical diagnosis, especially in the field and in resource-poor areas.

[Advances in quality research of Cinnamomum cassia].

Cinnamomum cassis is one of the commonly used traditional Chinese medicines in China. Its genuine producing areas distribute in Guangdong and Guangxi provinces. As an important edible herb and export variety of China, the quality control and internationalization of quality standards of C. cassis is extremely significant. In the recent years, with the development of the cinnamon industry, relevant academic research and the upgrade of the international standards, it is necessary to summarize the quality-related progress of C. cassis. In the present review, the germplasm resources, specific quality marker(Q-marker) and quality standards of C. cassis were summarized on the basis of published research during the last 10 years.

Development of a real-time reverse transcription recombinase polymerase amplification assay for rapid detection of spring viremia of carp virus.

Spring viremia of carp virus (SVCV) is a significant pathogenic agent that can cause large-scale outbreaks of spring viremia of carp (SVC) in many types of fish and bring huge economic losses to the aquaculture industry. A simple and convenient detection method is imperative for SVCV diagnosis. In this study, the real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and validated. Primers and probe targeting the conserved region of M gene were designed and applied to the real-time RT-RPA assay that performed at 39 °C for 20 min. The specificity analysis showed that no cross-reaction with other pathogenic viruses of fish was found, indicating appropriate specificity of the assay. In vitro transcribed RNA standards were used to estimate the sensitivity of the assay and the detection limit was 102copies/reaction. To further evaluate the assay, 65 clinical samples were tested using both real-time RT-RPA assay and real-time RT-PCR method. The same detection results were observed, suggesting the potential application of real-time RT-RPA assay in clinical sample detection. This is the first report on RPA assay for SVCV detection and this new developed assay would be useful in both laboratory and in the field for diagnosis of SVCV.

Rapid and sensitive real-time recombinase polymerase amplification for detection of Marek's disease virus.

Marek's disease (MD) is one of the most devastating diseases of poultry. It's caused by the highly infectious alphaherpesvirus MD virus serotype 1 (MDV-1). In this study, a rapid and easy-to-use assay based on recombinase polymerase amplification (RPA) was developed for MDV detection. Primer-probe sets targeting the highly conserved region of Meq gene were designed and applied to the RPA assay. The assay was carried out on a real-time thermostatic fluorescence detector at 39 °C for 20 min. As revealed by the results, no cross-reactions were found with the Newcastle disease virus (NDV), chicken infectious anemia virus (CAV), infectious bursal disease virus (IBDV), avian infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avain influenza virus (AIV), avian leucosis virus (ALV), avian reovirus (ARV), Marek's disease virus serotype 2 (MDV-2) and turkey herpes virus (HVT), indicating appropriate specificity of the assay. Plasmid DNA standards were used to determine the sensitivity of the assay and the detection limit was 102copies/µL. To further evaluate the clinical performance, 94 clinical samples were subjected to the RPA assay and 28 samples were tested MDV positive, suggesting that the real-time RPA assay was sufficient enough for clinical sample detection. Thus, a highly specific and sensitive real-time RPA assay was established and validated as a candidate for MDV diagnosis. Additionally, the portability of real-time RPA assay makes it suitable to be potentially applied in clinical diagnosis in the field, especially in resource-limited settings.

Development of a real-time loop-mediated isothermal amplification assay for detection of porcine circovirus 3.

BACKGROUND: Porcine circovirus type 3 (PCV3) is an emerging circovirus species, that has been reported in major pig-raising countries including the United States, China, South Korea, Brazil, Spain, and Poland. RESULTS: A real-time loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of porcine circovirus 3 (PCV3). The method had a detection limit of 1 × 101 copies/µL with no cross-reactions with classical swine fever virus (CSFV) C strain, foot-and-mouth disease virus (FMDV), porcine circovirus 2 (PCV2) LG vaccine strain, porcine epidemic diarrhoea virus (PEDV), porcine respiratory and reproductive syndrome virus (PRRSV), or pseudorabies virus (PRV). The PCV3 positive detection rate of 203 clinical samples for the real-time LAMP assay was 89.66% (182/203). CONCLUSIONS: The real-time LAMP assay is highly sensitive, and specific for use in epidemiological investigations of PCV3.

High-throughput Luminex xMAP assay for simultaneous detection of antibodies against rabbit hemorrhagic disease virus, Sendai virus and rabbit rotavirus.

Rabbits are widely used as models in biological research, and the pathogen status of rabbits used in studies can directly affect the results of experiments. Serological surveillance is the common monitoring method used in laboratory animals. A rapid, sensitive, and cost-effective high-throughput Luminex xMAP assay could be an attractive alternative to labor-intensive enzyme-linked immunosorbent assay (ELISA) methods. In this study, recombinant proteins from rabbit hemorrhagic disease virus and rabbit rotavirus and whole viral lysates of Sendai virus were used as coating antigens in an xMAP assay for the simultaneous detection of antibodies against these pathogens. The xMAP assay showed high specificity, with no cross-reaction with other pathogens. The coefficient of variation for intra-assay and inter-assay comparisons was less than 3% and 4%, respectively, indicating good repeatability and stability of the assay. The xMAP assay exhibited similar limits of detection for rabbit hemorrhagic virus and Sendai virus and was less sensitive for the detection of rabbit rotavirus when compared with commercial ELISA kits. A total of 52 clinical samples were tested simultaneously using both the xMAP assay and ELISA kits. The results obtained using these two methods were 100% coincident. In summary, the novel xMAP assay offers an alternative choice for rapid and sensitive high-throughput detection of antibodies in rabbit serum and can be used as a daily monitoring tool for laboratory animals.

Point-of-care diagnostic assay for rapid detection of porcine deltacoronavirus using the recombinase polymerase amplification method.

Porcine deltacoronavirus (PDCoV) has emerged and spread throughout the porcine industry in many countries over the last 6 years. PDCoV caused watery diarrhoea, vomiting and dehydration in newborn piglets. A sensitive diagnostic method would be beneficial to the prevention and control of PDCoV infection. Recombinase polymerase amplification (RPA) is an isothermal amplification method which has been widely used for virus detection. A probe-based reverse transcription RPA (RT-RPA) assay was developed for real-time detection of PDCoV. The amplification can be finished in 20 min and fluorescence monitoring was performed by a portable device. The lowest detection limit of the PDCoV RT-RPA assay was 100 copies of RNA molecules per reaction; moreover, the RT-RPA assay had no cross-reaction with other common swine viruses. The clinical performance of the RT-RPA assay was evaluated using 108 clinical samples (54 intestine specimens and 54 faecal swab specimens). The coincidence rate of the detection results for clinical samples between RT-RPA and RT-qPCR was 97.2%. In summary, the real-time RT-RPA assay offers a promising alternative to RT-qPCR for point-of-care detection of PDCoV.

The complete chloroplast genome sequence of Pohlia cruda (Hedw.) Lindb.

Pohlia is a genus with many taxonomic and systematic controversies. In this study, the complete chloroplast genome of Pohlia cruda (Bryales, Bryophyte) was sequenced by high-throughput sequencing technology and described. The complete chloroplast genome is 125,114 bp in length and has a quadripartite structure. The two inverted repeat (IR) regions are 9921 bp long and separated by a large single-copy (LSC) region of 86,727 bp and a small single-copy (SSC) region of 18,545 bp. Phylogenetic trees were constructed based on the complete chloroplast genome sequences of 10 bryophytes downloaded from GenBank and one acquired in this study.