The H7N9 virus mutated in 2017, resulting in new cases of highly pathogenic avian influenza (HPAI) H7N9 virus infection. H7N9 was found in a viraemic patient in Guangdong province, China. The present study aimed to clarify the pathogenic characteristics of HPAI H7N9. Virus was isolated from the plasma and sputum of the patient with HPAI H7N9. Liquid phase chip technology was used to detect the plasma cytokines from the infected patient and healthy controls. Mice were infected with strains A/Guangdong/GZ8H002/2017(H7N9) and A/Zhejiang/DTID-ZJU01/2013(H7N9) to observe the virus’s pathogenic characteristics. Serum and brain tissue were collected at 2, 4, and 6 days after infection. The viruses in serum and brain tissue were detected and isolated. The two strains were infected into A549 cells, exosomes were extracted, and virus genes in the exosomes were assessed. Live virus was isolated from the patient’s plasma. An acute cytokine storm was detected during the whole course of the disease. In animal experiments, A/Guangdong/GZ8H002/2017(H7N9) was more pathogenic than A/Zhejiang /DTID-ZJU01/2013(H7N9) and resulted in the death of mice. Live virus was isolated from infected mouse serum. Virus infection was also detected in the brain of mice. Under viral stress, A549 cells secreted exosomes containing the entire viral genome. The viraemic patient was confirmed to have an HPAI H7N9 infection. A/Guangdong/GZ8H002/2017(H7N9) showed significantly enhanced toxicity. Patient deaths might result from cytokine storms and brain infections. Extrapulmonary tissue infection might occur via the exosome pathway. The determined pathogenic characteristics of HPAI H7N9 will contribute to its future treatment. In February 2013, the avian influenza A H7N9 virus was identified as causing human infection, with high mortality and morbidity. To date, five waves of H7N9 infection have caused more than 1500 severe human infections and the mortality remains high, with more than 600 deaths  in ChinaIn a previous study of H5N1 virus infection, viraemia was detected in 82% of the fatal cases, but absent in nonfatal cases. High viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis. Individuals with detectable H5N1 RNA in their blood also had higher pharyngeal viral loads than those without evidence of H5N1 RNA, suggesting that the presence of viral RNA in blood reflects an overall high viral burden, which commonly leads to high mortality . In our Genetic changes to viruses might change their behaviour and pathogenic characteristics . We treaExosomes play an important role in viral infection . StudiesThe work was carried out in accordance with ethical guidelines of the Guangzhou Eighth People’s hospital . Sputum and plasma specimens were collected daily after admission. The patient’s plasma remaining after routine testing was stored in a bio-sample banker. The normal plasma were collected from 10 volunteers . The median age of these volunteers was 26 (interquartile range 23–28 year) year old. These volunteers came to the First Affiliated Hospital of Zhejiang University School of Medicine for regular physical examination. After the physical examination, these volunteers were confirmed healthy. The remaining plasma after routine testing was stored and used in our study.Female specific-pathogen-free (SPF) BALB/c mice, aged 6–8 weeks old, were purchased from the Shanghai Laboratory Animal Center, China. All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Zhejiang Province and the study was approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University . All experiments with H7N9 virus were performed in a biosafety level 3 laboratory approved by the First Affiliated Hospital, School of Medicine, Zhejiang University (Permit No. 2015-15).The Madin-Darby canine kidney cell line (MDCK) and A549 cell line  were obtained from the ATCC . The cell lines were propagated in growth medium containing Dulbecco’s modified Eagle’s medium  supplemented with 10% foetal bovine serum  at 37°C and 5% CO2. The A/Guangdong/GZ8H002/2017(H7N9) virus used in this study was isolated from a patient in Guangzhou, China, in 2017 (GenBank: MF455313- 455320). We isolated this virus from the patient’s plasma. The A/Zhejiang /DTID-ZJU01/2013(H7N9) virus was isolated from a patient in Zhejiang Province, China, in 2013 . Virus sThe viral RNA of the H7N9 virus was extracted using Trizol  method. The concentration, purity, and integrity of total viral RNA were detected using a NanoDrop 2000 instrument  and an Agilent 2100 Bioanalyzer . The sequencing library was constructed using TruSeq Stranded Total RNA with Ribo-zero Gold , and the process was conducted according to the manufacturer’s protocol. The library was constructed as follows: after digesting ribosomal RNA with Ribo-zero, the RNA was broken into fragments of about 350 nt using Fragment High Mix, and the sequencing library was constructed by synthesizing and purifying cDNA, terminal repair, adding poly-A tail, ligating sequencing connectors, and PCR amplification. After quality checking, the library was sequenced using an Illumina Hiseq Xten sequencer. The sequencing strategy generated 150 bp paired ends (PE150).Clean reads were obtained through data quality control and filtering of raw sequencing data using the NGSQC toolkit . Clean RThe TCID50 (Fifty percent tissue culture infective dose) assay was performed as described previously. The vir2. When the cell density reached 70–80%, the cells were washed three times with PBS to remove the bovine serum. The cells were then infected with the viruses (MOI=0.1). After the adsorption for 2 h, the cells were washed three times with PBS. DMEM supplemented with 1% foetal bovine albumin and 1 µg/ml L-l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) was added to the culture A549 cells for 48 h when culturing the ZJU01 strain. DMEM supplemented with 1% foetal bovine albumin was added to the culture A549 cells for 48 h while culturing the GZ8H002 strain. The virus culture supernatant was measured by the quantity of RNA and TCID50 titre.A549 cells were cultured in DMEM supplemented with 10% FBS at 37°C and 5% COAfter culture for 48 h, the cells were washed twice with PBS slightly. Cells were then fixed with 4% paraformaldehyde for 20 min. After washed twice with PBS slightly, cells were penetrated with 0.1% Triton X-100 for 20 min. Cells were again washed, blocked for 30 min with 3% bovine serum albumin  and incubated sequentially with 1:400 dilution of mouse anti-influenza A virus nucleoprotein antibody  for 1 h. After washing 5 times with PBS, the Alexa Fluor 488 conjugated goat anti-mouse IgG  with 1:500 dilution was used as the secondary antibody. After washing 5 times with PBS, DAPI  was used as the nuclear counterstain. After washing 5 times with PBS, the cells were observed under the fluorescence microscope.4 TCID50 A/Guangdong/GZ8H002/2017(H7N9) or the A/Zhejiang/ DTID-ZJU01/2013(H7N9) virus. Phosphate-buffered saline  of the same volume was given to the mice in the control group. Mice were observed for illness, weight loss, and death post-infection. Then, the mice were sacrificed, and their serum was collected at 2, 4, and 6 days post-infection (dpi) . The lung and brain were removed. Part of the organ was fixed in 10% buffered formalin, while the other part was used to detect virus levels using quantitative PCR. An H7N9 nucleic acid quantitative detection kit (Cat#Z-RR-0309-02) and Influenza virus A Real time RT-PCR kit (Cat#RR-0051-01) were purchased from Zhijiang biological technology Co., Ltd. Shanghai, China.Mice were inoculated intranasally with 50 µl 10g for 3 h in a Beckman Coulter Optima XPN-80 Ultracentrifuge . The pellet of virus was resuspended by 200 µl of PBS. The viruses were observed using a Tecnai Spirit 120 kv transmission electronic microscope to analyse their structure.The sera collected at 2, 4, and 6 dpi were used to isolate the virus. About 100 µl of serum was injected into the allantoic cavities of 9-day-old SPF embryonated chicken eggs. The embryonated chicken eggs were cultured in an incubator at 37°C for 72 h. The allantoic fluid was harvested and tested using an HA assay. Then 100ul serum from the 6 dpi infected mice was used to detect virus levels using quantitative PCR. Meantime, we collected 1.2 ml of serum from the 6 dpi infected mice and diluted in 40 ml of PBS. The dilution was subjected to ultra-centrifugation at 120000 × 2O2 in methanol. Sections were blocked for 1 h with 3% bovine serum albumin  in PBS and incubated sequentially overnight at 4°C with 1:200 dilution of polyclonal rabbit anti-H7N9 antibodies. Antibody binding was detected using EnVision System reagents . All slides were counterstained with haematoxylin.The amount of virus was evaluated using quantitative PCR. Organ tissue was prepared for haematoxylin eosin (HE) staining. Immunohistochemical (IHC) assays were also conducted. Paraffin sections of organs were de-waxed and then subjected to heat treatment in citrate buffer, and endogenous peroxidase activity was quenched using 0.3% HThe plasma of the patient was collected from the patient with HPAI H7N9 infection. Ten normal plasma samples were collected from the healthy volunteers. The cytokines and chemokines in human plasma was measured using the Bio-Plex Pro™ Human Cytokine Screening Panel, 48-Plex . Serum samples from the mice were detected using Bio-Plex Pro™ Mouse Cytokine Grp I Panel 23-Plex . The detection was based on the Bio-Plex 200 system (Bio-Rad) and performed according to the manufacturer instructions. g to remove the cells. Then 40 ml virus culture supernatant was obtained. The supernatant was then subjected to ultra-centrifugation at 120000 × g for 3 h in Beckman Coulter Optima XPN-80 Ultracentrifuge. The pelleted exosomes were resuspended in 200 µl of PBS. The exosomes were then observed using a Tecnai Spirit 120 kv transmission electronic microscope.The culture of the two strains of viruses in A549 cell model was described in the previous part. After 48 h of culture, the culture supernatant was collected by centrifugation at 500 ×To separate the virus from the exosomes, exosomes were purified using an Exosome-human CD63 Isolation kit . The exosomes were bound to the magnetic beads marked by CD63. The viruses were separated by washing with solution buffer. Then, 40 µl of magnetic beads were washed with 200 µl of isolation buffer (PBS with 0.1% BSA). The pre-enriched exosome solution was titrated with isolation buffer for the magnetic beads and mixed well. The beads were incubated overnight for 22 h at 4°C with mixing. The bead-bound exosomes were washed by adding 400 µl of isolation buffer and mixed gently by pipetting. The tube was placed on a magnet for 1 min and the supernatant was discarded. These steps were repeated three times. The purification of exosome was verified, with no H7N9 detected in supernatant (Cq > 38 was considered negative). The last supernatant was collected and the bead-bound exosomes were resuspended by 300 µl of isolation buffer. The identification of H7N9 genes in the exosome was performed using an H7N9 nucleic acid quantitative detection kit. Then, 100 µl of bead-bound exosomes solution was injected to the allantoic cavities of 9-day-old SPF embryonated chicken eggs and cultured at 37°C for 72 h. The allantoic fluid was harvested and tested using an HA assay.P-values < 0.05 were considered statistically significant.Statistical analyses of data for weight and survival rate were performed using Graphpad Prism 5 . Statistical analyses of the data for cytokines and chemokines were conducted using the Mann–Whitney test. Statistical analyses of the data for H7N9 gene expression in A549, embryonated chicken egg and mouse serum were conducted using a one-way analysis of variance (ANOVA) test with Tukey’s multiple comparison test. Statistical analyse of the data for H7N9 gene expression in exosome-enclosed viral particle was conducted using paired t test. Statistical analyses of the data for H7N9 gene expression in the brain and lung leachates were conducted using a two-way ANOVA test with Bonferroni post-tests. The one-way analysis of variance (ANOVA) test with Tukey’s multiple comparison test was used to compare the TICD50 titre of H7N9 virus isolated from the exosomes. The values are presented as means ± SD for the indicated sample sizes. The patient suffered a cytokine storm in the hospital. Forty-eight cytokines and chemokines were tested. Among them, the levels of 32 cytokines and chemokines including IL-1ra, IL-6, IP-10, IL-10, IFN-γ, MCP-3, IL-18, HGF, MCP-1, MIG, IL-2Rα, IL-12(p40), IL-8, MIP-1α, PDGF-BB, G-CSF, CTACK, MIF, SCF, HGF, IL-16, SCGF-β, RANTES, TNF-α, FGF basic, IL-2, IL-17, MIF, MIP-1β, SCF, SDF-1α, and TRAIL were elevated in the patient. The levels of ten cytokines and chemokines including MIG, IL-1ra, IL-6, IP-10, IL-10, IFN-γ, MCP-3, IL-18, HGF, MCP-1 were dramatically higher in this patient  comparedThe HPAI virus could be isolated from both the sputum and the plasma. Deep sequencing of the HPAI virus isolated from the sputum and the plasma were performed. The genome of ZJU01 strain was used as the reference genome. A circos diagram was constructed to present the mutations in the different genes . In the circos diagram, the innermost circle represents the virus isolated from the plasma. The other circles represent the virus isolated from the sputum collected at different times. The HPAI virus isolated from the sputum (three sputum samples from the same patient) and the plasma were highly similar. There were only rare mutations between them. However, the genes of the HPAI virus had many mutation in the eight viral genes compared with ZJU01 strain A.FigureWe further compared the replication ability of these two virus. When cultured in embryonated chicken eggs, the replication ability of A/Guangdong/GZ8H002/2017(H7N9) virus was better than that of A/Zhejiang /DTID-ZJU01/2013(H7N9). The quantity of RNA and the TCID50 titre of A/Guangdong/GZ8H002/2017(H7N9) was higher than those of A/Zhejiang /DTID-ZJU01/2013(H7N9) B,C. WhenTo determine whether the A/Guangdong/GZ8H002/2017(H7N9) strain had acquired any key molecular substitutions associated with increased virulence and transmissibility in mammals, or with antiviral drug resistance, we performed deep sequencing of the HPAI H7N9 virus. As shown in We observed illness, weight loss, and death in the mice after H7N9 challenge. Various amounts of weight change were noted in all three groups A. The miWe isolated the H7N9 virus from the mouse serum. In the A/Guangdong/GZ8H002/2017(H7N9) infection mice, the live virus was detected persistently in the serum until death . The serWe collected the serum of mice infected with A/Zhejiang/DTID-ZJU01/2013(H7N9) and A/Guangdong/GZ8H002/2017(H7N9) in early (2 dpi) and late stages (6 dpi) of infection and detected the serum cytokine levels. In the early stage of infection, IL-6, G-CSF, and IL-13 levels were high in the A/Zhejiang/DTID-ZJU01/2013(H7N9) and A/Guangdong/GZ8H002/2017(H7N9) infection groups E-G. In tThe histopathology of organ tissues was evaluated using HE staining. The extent and characteristics of the lesions varied among the groups. The mice infected with A/Zhejiang/DTID-ZJU01/2013(H7N9) had multifocal interstitial inflammatory hyperaemia and exudative pathological changes in the lung, with larger lesions in the lung tissue and fusion of multiple patchy lesions. These histopathological changes were severe at 2 dpi. In the following days, the pathological changes of the lung tissue persisted, but gradually improved. At 6 dpi, the pathological change became moderate. This was consistent with the improvement in the mice’s general health A,B. The High levels of the H7 virus gene were detected persistently in the lungs of the H7N9-infected mice C. At 2 dIn both infection groups, HE staining of brains presented inflammatory cell infiltration B, E. In We also detected the expression of the H7 gene in brain leachate C. The H7The TCID50 of brain leachate was only detected at 6 dpi in the A/Guangdong/GZ8H002/2017(H7N9)-infected mice D. In theThe exosomes were enriched using ultra-centrifugation. The morphology of the exosome was viewed using TEM. The typical shape of exosomes was observed in the different groups. The shape and size of the exosome varied from 50 to 200 nm. The culture supernatant from A549 cell model without virus infection was observed using transmission electronic microscope. The exosomes were showed by blue arrow A. The cuAfter purification, the H7N9 virus genes in the exosomes were detected using quantitative PCR. The exosome-enclosed viral particle is a very small part compared to the total viral particle. The H7 gene content of total viral particle and exosome-enclosed viral particle were presented in HPAI H7N9 virus infection starts in the respiratory tract. After infection, HPAI H7N9 virus replicates in the lung. Meanwhile, the patient suffered from persistent viraemia and a cytokine storm was observed his plasma. Live H7N9 virus was detected the patient’s blood. These phenomena were confirmed using animal experiments. How is the virus transmitted from the lung to the blood? One possible explanation is that inflammation leads to the disruption of the lung-blood barrier. Another possibility is that the virus is released into the blood through via exosomes. When the H7N9 virus exists in the blood, the virus might cause extrapulmonary infection. We speculated that the virus could infect the brain, after observing this phenomenon in the mouse model . HoweverIn the present study, HPAI H7N9 infection of a patient proved to be deadly. This patient presented with viraemia and died 3 days after admission. Whether there is live virus in the blood and whether it could lead to extrapulmonary infection have been two important questions. Previous studies detected viral RNA from LPAI H7N9, H5N1, and H1N1 infections ,8. H5N1 In the previous studies of H5N1 and pandemic 2009 H1N1 virus infection, viraemia was commonly existed in the fatal cases, but was absent in nonfatal cases . In prevIn our previous study, we observed that the levels of 34 of the 48 cytokines and chemokines tested were significantly elevated in plasma samples from patients infected with LPAI H7N9. We further found that the levels of MIF, SCF, MCP-1, HGF, and SCGF-β were highly positively correlated to disease severity. In addition, the profile of mediators MIF, SCF, MCP-1, HGF, SCGF-β, IP-10, IL-18, and IFN-γ is an independent outcome predictor . Cytokinin vitro study, avian H7N9 virus could effectively infect human brain cells. Meanwhile, the virus could be transcribed, replicate its viral genome, and generate infectious progeny virus [Influenza virus infection of the respiratory tract is associated with a range of neurological complications. The incidence of severe pandemic H1N1 influenza-associated neurological complications was estimated to be 1.2 per 1,00,000 persons among children and Asian Pacific patients ,33. The ny virus . In the However, we must say that we haven’t fully revealed the potential mechanism behind this feature of the 2017 H7N9 strain. But we observed the GZ8H002 strain might have acquired the possible key molecular substitutions associated with increased virulence and transmissibility in mammals. As we know, the presence of KRTA insertion in the cleavage sites enhanced the extrapulmonary infection ability and virulence of HPAI virus ,39. ThisExosomes are cellular secreted vesicles that are free to enter cells in a receptor-independent manner. Exosomes have been found to be closely associated with viral infection , and canIn summary, the viraemic patient was confirmed to be infected with HPAI H7N9. Cytokine storms and potential brain infections might be the cause of patient death. Exosomes might be the pathway that leads to infection in extrapulmonary tissues.


Citrus species in which the virus primarily infects phloem-associated cells. Despite all these difficulties, there have been many instances when research on CTV has resulted in the discovery of novel aspects and phenomena in virus–virus and virus–host interactions and has allowed to generate reagents, which may not be amenable with small, less complex viruses. This Review discusses five exciting and important facts about this virus.Citrus tristeza virus (CTV), a member of the family Closteroviridae, represents one of the most intricate viruses with an overwhelmingly complex biology. From a standpoint of economic importance, CTV is regarded as the most destructive viral pathogen of citrus. During the past century, virus-induced epidemics of quick decline , a diseaCTV possesses a 19.3 kilobase (kb) positive-stranded RNA genome, which is nearly twice as large as the average sized RNA virus genome of 10 kb . BesidesMost of the CTV proteins play multiple roles in the virus infection cycle, which is in agreement with often found multifunctional nature of viral proteins. Yet, one of them, a unique nonconserved p33, appears to be the champion when it comes to the functions it performs. The research demonstrated that p33 is a membrane-associated protein, and its membrane association confers virus ability to extend the host range , 8. The During infection, CTV produces a plethora of different RNA species. Those include the genomic RNA and its complementary negative-stranded copy, a nested set of 3’-coterminal sgRNAs that serve as mRNAs for translation of the internal and 3’-end genes and their corresponding negative-stranded complements along with the 5’-terminal positive-stranded sgRNAs, which are generated as a result of promoting or terminating activity of the same sgRNA controller elements CEs; ; Fig 1D)Fig 1D). CTV has numerous variants, which could be grouped in at least seven major strains. Strains of CTV are defined as phylogenetically distinct lineages, which are classified based upon analysis of the viral genomic sequences . IndividThe explanation for coexistence of multiple strains of CTV in a host came from research on CTV SIE. SIE, a phenomenon in which a primary virus infection prevents a secondary infection with the same or closely related virus, has been observed with viruses in various systems, including important pathogens of humans, animals, and plants. With CTV, SIE only occurs between virus variants of the same strain, while variants belonging to different strains do not exclude each other, permitting formation of multistrain populations in the same host  Fig 1F . With vaKnowledge obtained from the research on CTV SIE can be translated into practical applications that would allow protection of citrus trees in the field from aggressive CTV variants. One such application is crossprotection or preimmunization, a purposeful inoculation of trees with mild virus variants. This strategy has been widely practiced in many citrus-growing regions, yet with low success due to the lack of understanding of the mechanisms behind a mild variant-mediated protection . KnowingTraditionally, CTV has been considered as a phloem-limited virus , 5. On tA few years ago, CTV was developed into a vector for transient expression of sequences of interest  in citrus trees, which opened new avenues for managing citrus and, possibly, other perennial crops . Upon inLiberibacter asiaticus, the disease causal agent, or the insects that transmit the bacterium. Importantly, although expected to work for a relatively long term, the modified viruses will eventually lose the inserted sequences such that those vectors would not add anything permanently to the environment.Several important traits make the CTV-based vector an attractive tool for crop management. One of them is its unprecedented stability: the modified virus has retained and expressed a foreign insert in planta for years, which compares to days or weeks for most other vectors engineered based on smaller plant viruses. Such a feature of the CTV vector correlates with an unusual intrinsic stability of the CTV consensus genome and slow evolutionary rate shown for this virus . The CTV


Urine cell-free DNA (cfDNA) is a valuable non-invasive biomarker with broad potential clinical applications, but there is no consensus on its optimal pre-analytical methodology, including the DNA extraction step. Due to its short length (majority of fragments <100 bp) and low concentration (ng/mL), urine cfDNA is not efficiently recovered by conventional silica-based extraction methods. To maximize sensitivity of urine cfDNA assays, we developed an ultrasensitive hybridization method that uses sequence-specific oligonucleotide capture probes immobilized on magnetic beads to improve extraction of short cfDNA from large-volume urine samples. Our hybridization method recovers near 100% (95% CI: 82.6–117.6%) of target-specific DNA from 10 mL urine, independent of fragment length (25–150 bp), and has a limit of detection of ≤5 copies of double-stranded DNA (0.5 copies/mL). Pairing hybridization with an ultrashort qPCR design, we can efficiently capture and amplify fragments as short as 25 bp. Our method enables amplification of cfDNA from 10 mL urine in a single qPCR well, tolerates variation in sample composition, and effectively removes non-target DNA. Our hybridization protocol improves upon both existing silica-based urine cfDNA extraction methods and previous hybridization-based sample preparation protocols. Two key innovations contribute to the strong performance of our method: a two-probe system enabling recovery of both strands of double-stranded DNA and dual biotinylated capture probes, which ensure consistent, high recovery by facilitating optimal probe density on the bead surface, improving thermostability of the probe-bead linkage, and eliminating interference by endogenous biotin. We originally designed the hybridization method for tuberculosis diagnosis from urine cfDNA, but expect that it will be versatile across urine cfDNA targets, and may be useful for other cfDNA sample types and applications beyond cfDNA. To make our hybridization method accessible to new users, we present a detailed protocol and straightforward guidelines for designing new capture probes. Urine cell-free DNA (cfDNA) is generated from transrenal excretion of circulating cfDNA, as well as from local degradation of cells shed along the urogenital tract . It is aConventional silica-based DNA extraction methods are not suitable for urine cfDNA, which is dilute and extensively fragmented. While plasma cfDNA has a peak fragment length of approximately 167 bp, reflecting protection of histone-associated DNA within nucleosomes , urine cAs further evidence of the importance of targeting short fragments, decreasing PCR amplicon length has been shown to increase both clinical sensitivity and the detected concentration of urine cfDNA ,21,22. DThe ability to detect short fragments is particularly important given the low total concentration of cfDNA in urine (<1–200 ng/mL) ,23 and pWe aimed to develop an improved method for extraction of short cfDNA from urine. Our goal was to improve purification and detection of known target sequences for diagnostic applications, rather than to extract total urine cfDNA. We identified sequence-specific hybridization capture as a technique likely to perform well for short, dilute urine cfDNA because it should be agnostic to DNA fragment length and concentration. Sequence-specific purification uses oligonucleotide probes that are complementary to the sequence(s) of interest to capture target-specific nucleic acids via hybridization. The capture probes are immobilized on a substrate (commonly magnetic beads) to allow for isolation and concentration of the target-specific nucleic acid. Generally, there are two hybridization capture formats distinguished by the order of the target-probe hybridization and probe immobilization steps. The first, referred to here as solution-based hybridization, involves hybridization of biotinylated probes to target in solution, followed by capture of target-probe duplexes on streptavidin-coated magnetic beads . The sece.g., targeted enrichment of sequencing libraries). From this survey of previous applications of hybridization capture for sample preparation, the versatility and potential high sensitivity and specificity of the technique are clear. The best reported analytical sensitivities are in the range of 5–100 copies, genome equivalents, or colony forming units, with a maximum sample input of 1 mL. Purification efficiencies, when reported, were variable (<10% to 97%) but suggest the potential for high recovery if conditions are optimized. Unfortunately, many previous studies did not report detailed protocols or characterization of their methods , making direct comparison difficult. Non-optimal probe concentrations, bead volumes, and probe:bead ratios may have resulted in differences in performance across protocols. Reports on the performance of solution-based versus surface-based hybridization are inconsistent, with no consensus on when one approach may be preferred to the other.Hybridization capture has been previously used for sequence-specific purification of nucleic acids directly from a variety of sample types, as summarized in In previous implementations, hybridization capture was used primarily to isolate target DNA from an excess of non-target DNA, which can inhibit downstream amplification and reduce sensitivity, particularly for dilute targets in an excess of non-target DNA ,56,58. FHere, we report the development and optimization of an ultrasensitive hybridization method that uses dual biotinylated sequence-specific probes immobilized on streptavidin-coated magnetic beads to capture, concentrate, and purify target-specific cfDNA fragments from 10 mL urine samples. Our hybridization method is intended for sample preparation prior to qPCR-based detection of known target sequences, such as in the case of pathogen detection; it is not suitable for applications requiring purification of total cfDNA prior to sequencing. We have identified key parameters affecting hybridization efficiency and implemented designs to ensure consistent, high percent recovery. We report full characterization of the analytical performance of our hybridization capture method and describe two key innovations that contribute to its unprecedented sensitivity: dual biotinylated capture probes and a two-probe system for recovery of both strands of double-stranded DNA (dsDNA). Our hybridization method improves upon the analytical performance of both existing silica-based urine cfDNA extraction methods and hybridization capture protocols, including our own previously-reported 1 mL solution-based hybridization assay . Althoughttp://dx.doi.org/10.17504/protocols.io.bep4jdqw). The protocol below was used for analytical characterization of the hybridization capture method, with experimental details and any modifications for individual experiments noted in the following subsections.A full step-by-step hybridization capture protocol is posted on protocols.io  with 500 μL 0.5 M EDTA and 100 μL 1M Tris-HCl pH 8.Collect urine sample in a sterile container.Immediately after urine collection, add 10 mL of urine to each prepared 15 mL tube and mix by inversion. The final concentration will be 25 mM EDTA and 10 mM Tris-HCl.Freeze at -80°C until analysis.Immediately before analysis, thaw urine at 37°C and mix by inversion.Optionally, test with Fisherbrand 10-SG Urine Reagent Strips .Centrifuge urine for 5 minutes at 8000g to pellet cell debris.Transfer cell-free urine supernatant to new 15 mL DNA LoBind tube (Eppendorf).Immobilize capture probes on magnetic beadsVortex Dynabeads MyOne Streptavidin C1 (Thermo Fisher) for 30 seconds to ensure that beads are evenly dispersed in solution.Pipette beads into a 1.5 mL DNA LoBind tube (Eppendorf). Prepare 50 μL (0.5 mg) beads per 10 mL urine sample to be analyzed.Place beads on magnetic rack for 1 minute; remove and discard supernatant.Add an equal volume of high salt wash buffer , vortex for 5 seconds, and briefly spin down.Repeat twice for a total of three washes.Wash beads three times with an equal volume of high salt wash buffer .Resuspend beads in an equal volume of high salt wash buffer .Pre-mix dual biotinylated capture probes BP1 and BP2  in TLE tAdd pre-mixed dual biotinylated capture probes BP1 and BP2 to beads and immediately vortex for 5 seconds. Use 25 pmol of each probe per 50 μL beads (0.5 μL of pre-mixed stock with 50 μM of each probe). If using only a single probe, use 50 pmol per 50 μL beads.Rotate for 15 minutes at room temperature to immobilize probes on beads.Briefly spin down.Place beads on magnetic rack for 1 minute; remove and discard supernatant.Add an equal volume of high salt wash buffer , vortex for 5 seconds, and briefly spin down.Repeat twice for a total of three washes.Wash beads three times with an equal volume of high salt wash buffer .Resuspend beads in an equal volume high salt wash buffer.Capture target-specific urine cfDNA by hybridizationAdd 2.5 mL 5 M NaCl , 127 μL 10% Tween-20  and 50 μL prepared beads to each 10 mL urine sample. If spiking in positive control , add it Mix well by inversion.Denature for 15 minutes in dry bath with 15 mL tube block preheated to 120°C (urine temperature should reach >90°C).Rotate for 30 minutes at room temperature to hybridize target cfDNA to capture probes.Wash to remove urine inhibitors and non-target DNACentrifuge for 5 minutes at 5000g to pellet beads.Remove and discard all but ~1 mL supernatant using 10 mL serological pipette.Resuspend beads in remaining supernatant and transfer to 1.5 mL DNA LoBind tube (Eppendorf).Place on magnetic rack for 1 minute. Remove and discard supernatant, then remove tube from magnetic rack.Add 1 mL high salt wash buffer  and wash by inverting 10–20 times, or until no bead aggregate is left on tube wall. Do not vortex. Spin down briefly.Place on magnetic rack for 1 minute. Remove and discard supernatant, then remove tube from magnetic rack.Add 1 mL high salt wash buffer  and wash by inverting 10–20 times, or until no bead aggregate is left on tube wall. Do not vortex. Spin down briefly.Place on magnetic rack for 1 minute. Remove and discard supernatant, then remove tube from magnetic rack.Add 1 mL low salt wash buffer  and wash by inverting 10–20 times, or until no bead aggregate is left on tube wall. Do not vortex. Spin down briefly.Place on magnetic rack for 1 minute. Remove and discard supernatant.Spin down again, place on magnetic rack, and remove as much liquid as possible using P20 pipette.Elute purified target cfDNAAdd 20 μL freshly-prepared 20 mM NaOH, vortex for 5 seconds, and spin down briefly.Place on magnetic rack. Transfer as much supernatant as possible  directly to qPCR well or to new DNA LoBind tube (Eppendorf). This contains purified target cfDNA. Avoid transferring any beads to qPCR.Partially neutralize with 3.5 μL 100 mM HCl. The qPCR buffer will adjust to final pH, and tolerates slightly basic pH better than acidic pH.Proceed directly to qPCR.Quantify by qPCR4, 20 mM (NH4)2SO4, 2 mM MgSO4, 5% glycerol, 5% DMSO, 0.06% IGEPAL CA-630, 0.05% Tween-20, pH 9.2), 0.8 mM dNTPs (NEB), 0.4X EvaGreen , 200 nM forward primer  from each 10 mL urine sample in a single qPCR well. Each 50 μL reaction should contain 1.25 U OneTaq Hot Start DNA Polymerase , 1X NEB OneTaq GC Reaction Buffer  using an initial denaturation of 94°C for 3 min followed by 45 amplification cycles .Conduct post-amplification melt analysis from 65°C to 95°C in 0.5°C increments every 5 seconds.q values at a threshold of 500 RFU, calculate recovered copies using a standard curve  run for each experiment, and verify that melting temperature (Tm) matches that of expected amplicon.Determine Cm) to match that of the forward primer and increased the qPCR annealing temperature to 64°C . Participants gave written consent. For all experiments, the total capture probe concentration was kept constant at 50 pmol probe per 50 μL beads. For experiments targeting dsDNA (positive control and reverse complement), both probes BP1 and BP2 were used (25 pmol BP1 and 25 pmol BP2 per 50 μL beads) unless otherwise specified. For experiments targeting single-stranded DNA (ssDNA) (positive control only), only probe BP1 was used (50 pmol per 50 μL beads) unless otherwise specified. Some experiments used an older version of the reverse primer without LNA modifications and a lower qPCR annealing temperature of 58°C. Using these conditions, we occasionally observed late, probe-independent nonspecific amplification of residual human genomic DNA see . We upda3 copies of 50 bp dsDNA  of 50 bp dsDNA into 10 mL urine, extracting by hybridization, and amplifying by qPCR (n = 3). Representative calibration curves for the qPCR standards were generated by adding 0, 5, 10, 102, 103, 104, or 105 copies of 50 bp dsDNA directly into qPCR. The limit of detection was verified by spiking 0 or 5 copies (0.5 copies/mL) of 50 bp dsDNA into 10 mL urine, extracting by hybridization, and amplifying by qPCR .To determine the purification efficiency, 10bp dsDNA  were spi3 copies of 25, 50, 80, or 150 nt ssDNA , non-target DNA , and salt  conditions, extracted by hybridization, and amplified by qPCR using the reverse primer without LNA modifications (n = 1).To test the effects of variations expected in clinical urine samples, 103 copies of 50 nt ssDNA  . Amplification was carried out using the reverse primer without LNA modifications. An increase in quantification cycle (Cq) was used to indicate qPCR inhibition.To test for qPCR inhibition, eluate extracted from 10 mL urine (no added target) was spiked into qPCR containing 10To compare recovery of dsDNA using the one and two probe systems, 1000 copies of 50 bp dsDNA were spiked into 10 mL urine, extracted by hybridization, and amplified by qPCR. For the single probe system, 50 pmol of capture probe (BP1 or BP2) was used per 50 μL beads . For the two probe system, 25 pmol of each probe (BP1 and BP2) was used per 50 μL beads . To confirm that introduction of a second probe had no effect on the recovery of the opposite probe, 1000 copies of 50 nt ssDNA (positive control or reverse complement) were spiked into 10 mL urine, extracted by hybridization (50 pmol BP1 or BP2 or 25 pmol each of BP1 and BP2), and amplified by qPCR . To determine if re-hybridization of complementary target strands affected recovery, 1000 copies of 50 nt ssDNA or 50 bp dsDNA were spiked into 10 mL urine, extracted by hybridization (50 pmol BP1 or 50 pmol BP2), and amplified by qPCR . For these experiments, the reverse primer without LNA modifications was used.3 copies of 50 nt ssDNA were spiked into 10 mL urine, extracted by hybridization, and amplified by qPCR (n = 3 independent experiments processed on separate days). To test thermostability, beads were functionalized with 50 pmol dual biotinylated BP1 or the analogous single biotinylated probe per 50 μL beads and used to extract 103 copies of 50 nt ssDNA spiked into 10 mL urine. Beads were either added to the sample prior to denaturation at >90°C for 15 minutes, or after the sample was denatured and cooled to room temperature (n = 3 independent experiments processed on separate days). To test tolerance to free biotin, beads were functionalized with 50 pmol dual biotinylated BP1 or the analogous single biotinylated probe per 50 μL beads. Before hybridization, functionalized beads were incubated for 15 minutes at room temperature with or without 1 μM free D-biotin and washed three times with high salt wash buffer . Beads were then used to extract 103 copies of 50 nt ssDNA spiked into 10 mL urine (n = 3 independent experiments processed on separate days). For these experiments, the reverse primer without LNA modifications was used.To determine the dependence on probe concentration during immobilization, beads were functionalized with 5, 25, 50, 125, 250, or 500 pmol dual biotinylated BP1 or the analogous single biotinylated probe  per 50 μM. tuberculosis insertion element IS6110) to non-target probes (targeting the tuberculosis direct repeat (DR) region: 5’-/52-Bio/TCAGACCCAAAACCCCAAAAAAAAAAAAAAAAAAAA/3SPC3-3’ and 5’-/52-Bio/TCCGTCCCCTCTCGGAAAAAAAAAAAAAAAAAAAA/3SPC3-3’) was varied while keeping the total probe concentration constant at 50 pmol per 50 μL beads. For each condition, 103 copies of 50 bp dsDNA were spiked into 10 mL urine, extracted by hybridization, and amplified by qPCR . Conditions tested represented one double-stranded target (100:0 ratio of IS6110 probes to DR probes), two double-stranded targets (50:50), five double-stranded targets (20:80), and ten double-stranded targets (10:90). DR template DNA was also spiked in each condition to account for any effects due to binding of other targets .To demonstrate the feasibility of multiplexing with more than two probes, the ratio of target-specific probes BP1 and BP2 , conditions were compared using one-way ANOVA with Tukey’s honestly significant difference post hoc test. Statistical analysis was conducted using GraphPad Prism v8.1.2  with a significance level of 0.05.When appropriate (The hybridization capture probe design is shown in An overview of the hybridization capture procedure is given in Our hybridization capture method recovers 100.1% (95% CI: 82.6–117.6%) of DNA spiked into 10 mL urine . The calCritically for urine cfDNA, which is made up primarily of short fragments (majority <100 bp) ,15–17, oHybridization capture is expected to perform well across varied clinical urine samples due to the insensitivity of DNA hybridization to properties that vary in urine samples. Specifically, we have demonstrated tolerance to a wide range of pH (5–8), non-target DNA 0–10 μg), and salt (0–500 mM) conditions . Particu0 μg, andOur hybridization capture protocol also efficiently removes qPCR inhibitors in urine that may affect downstream amplification, enabling amplification of cfDNA from 10 mL urine in a single qPCR well without inhibition . AlthougBecause hybridization targets single-stranded DNA, it typically only captures half of available target molecules  compared to non-specific extraction methods such as silica adsorption that capture both strands of double-stranded DNA. To overcome this limitation, we designed two capture probes, one complementary to each strand of the dsDNA target . As showDual biotinylated probes, which have two biotin molecules in series at the 5’ end of the probes, are a key design element of our hybridization protocol that help ensure its high sensitivity and robust performance. As shown in 12 probes/cm2 [12 probes/cm2 (370–526 Å2/probe) on the bead surface, which is within the optimal range for rapid, efficient hybridization. Importantly, heterogeneity of probe distribution across the surface can also lead to sub-optimal recovery [Probe density has a significant, well-documented impact on the extent of DNA hybridization on surfaces, due to both thermodynamic and kinetic effects ,62. If tobes/cm2 ,63. We srecovery . Dual birecovery . The autrecovery .m of thermally-induced denaturation of streptavidin from 75°C to 112°C [In addition to moderating probe density, the dual biotin modification is advantageous because it increases thermostability of the bead-probe linkage. Beads functionalized with dual biotinylated probes maintained recovery after heating at >90°C for 15 minutes, while beads functionalized with single biotinylated probes lost all function after heating . Dual bito 112°C . The incto 112°C . Dual bito 112°C ,69 and eto 112°C ,70. For to 112°C .Saturating unbound streptavidin with biotin after probe immobilization may achieve a similar improvement in thermostability, but single biotinylated probes are negatively affected by the presence of high biotin concentrations. Although the streptavidin-biotin association is strong, some dissociation does occur . In the We have previously reported a solution-based hybridization assay, which had similar performance characteristics (73–84% recovery of 25–150 bp DNA) to the surface-based assay described here, but was limited to a 1 mL sample volume . BecauseBased on our experience, we generally recommend surface-based hybridization over solution-based hybridization, particularly when large sample volumes are required. Surface-based hybridization with dual biotinylated capture probes eliminates the risk of biotin interference while remaining cost-effective for large sample volumes, while solution-based hybridization may be compromised by high endogenous biotin concentrations and is more expensive. Assay time and percent recovery using the two approaches are comparable, as long as conditions are optimized. Specifically, optimal probe density is critical for success with surface-based hybridization, as discussed above.M. tuberculosis that had >90% recovery and ≤1 copy/mL sensitivity.Designing capture probes for new targets is straightforward. To reduce target footprint and minimize design effort, we recommend using truncated versions of the qPCR primers see . This ap4 used here). If amplification inhibition is observed, we suggest supplementing the qPCR buffer with additional Tris-HCl. To easily identify contamination and prevent false positives during clinical testing, we also recommend adding a 10–20 bp spacer region between the primer binding sites on your target of interest to design a synthetic positive control that can be distinguished from the native target of interest by post-amplification melt analysis  to maximize the fraction of cfDNA fragments that contain both primer sites and can be successfully amplified. Because the exact eluate volume varies slightly across samples, we found that partial neutralization leaving the eluate slightly basic had the least effect on downstream amplification. In optimization experiments where we controlled the eluate pH, the qPCR tolerated slightly basic eluate better than slightly acidic eluate. For new targets, we recommend using qPCR conditions with high buffering capacity  of a region within the target sequence while maintaining specificity.The capture probe sequences should be truncated versions of the primer sequences, with bases removed from the 5’ end to reach lengths of 15–20 nt long and melting temperatures of ~60°C at a concentration of 2 nM probe in 1 mM NaCl. A dual biotin modification and 20 nt poly(A) spacer should be added to the 5’ ends, and a C3 spacer should be added to the 3’ ends.i.e., the minimum concentration per probe) that maintains high recovery, we varied the ratio of target to non-target probes while keeping the total probe concentration constant  or RNA (any length). The results of previous studies , howeverOne area where our protocol may require modification is for sample types with high protein concentrations, such as plasma or sputum. If the magnetic beads are added prior to denaturation, according to the current protocol, it is likely that as proteins denature, they will adsorb to the bead surface and impact bead behavior. We have observed that beads become more likely to aggregate after heating in urine samples with proteinuria or blood present, resulting in less effective collection of beads on the magnet during wash steps and a tendency for beads to transfer to qPCR during the elution step. We are currently working on strategies, such as the addition of a Proteinase K digestion step, to mitigate bead loss in this situation. Another limitation resulting from the sequence-specific nature of hybridization capture is that it is designed for isolation and detection of specific target sequences, which will be most relevant for pathogen detection. Our hybridization capture method cannot be used to purify total DNA and is thus not appropriate for next-generation sequencing applications. Hybridization capture can, however, be multiplexed and ongoing work in our lab aims to develop a multiplexed system by adding capture probes to target multiple genomic regions. We have demonstrated feasibility of including multiple capture probes, showing capacity for up to 10 probes with only a minor reduction in recovery . AdditioTo increase the diagnostic sensitivity of cfDNA detection in urine, we aimed to develop an improved sample preparation method for short DNA fragments. We selected hybridization capture as a DNA extraction technique likely to perform well for short, dilute cfDNA. To enable successful detection of low concentrations of target-specific cfDNA in clinical urine samples, we pushed the limits of hybridization capture to achieve maximum analytical sensitivity and accommodate large-volume samples. Our final protocol improves upon both alternate urine cfDNA extraction methods  and prevS1 Fig2, 103, 104, 105 copies) are shown in grey and the NTCs (0 copies) are shown in red. The line at 500 RFU indicates the baseline threshold at which the threshold cycles were determined. Despite the variation in calculated percent recovery caused by minor differences in the standard curves, the 1000 copy hybridization output visually overlaps with the 1000 copy PCR standard across all three experiments, indicating near complete recovery of DNA by hybridization. We run a new qPCR standard curve for every experiment, so while calculated percent recovery may diverge from 100% for technical replicates from an individual experiment, we expect the calculated percent recovery across multiple independent experiments to center around 100%, as seen in e.g., In some cases, the calculated percent recovery of our hybridization assay may be >100% due to expected variations when quantifying by a qPCR standard curve. The relationship between qPCR threshold cycle and calculated starting quantity is exponential, so a small change in threshold cycle  can result in a large change in calculated starting quantity, and therefore percent recovery. Given here are the qPCR amplification curves for three example experiments with 70%, 99%, and 127% calculated recovery. The experimental samples (1000 copies extracted by hybridization) are shown as green curves with hollow circles. The qPCR standards Click here for additional data file.S2 Fig1000 copies of positive control target were extracted from 10 mL pooled urine with hybridization at room temperature or 45°C for 30 min or 60 min .(TIF)Click here for additional data file.S3 Fig3 copies 50 bp dsDNA input).Without additional assay optimization, multiplexed capture of up to 5 dsDNA targets or 10 ssDNA targets is possible with only moderate reduction in recovery compared to single-plex capture. To represent conditions expected during multiplexed capture, the ratio of target-specific probes to non-target probes was varied  while keeping the total probe concentration constant (50 pmol per 50 μL beads) Click here for additional data file.S1 Text(A) Primer design and optimization of PCR annealing temperature to encourage specific amplification. (B) Addition of spacer region to synthetic positive control to easily identify contamination and prevent false positives during clinical testing.(PDF)Click here for additional data file.


Gene expression is achieved by enzymes as RNA polymerases that translocate along nucleic acids with steps as small as a single base pair, i.e., 0.34 nm for DNA. Deciphering the complex biochemical pathway that describes the activity of such enzymes requires an exquisite spatiotemporal resolution. Magnetic tweezers are a powerful single molecule force spectroscopy technique that uses a camera-based detection to enable the simultaneous observation of hundreds of nucleic acid tethered magnetic beads at a constant force with subnanometer resolution . High spatiotemporal resolution magnetic tweezers have recently been reported [3–5]. We present data acquired using a bespoke magnetic tweezers instrument that is able to perform either in high throughput or at high resolution. The data reports on the best achievable resolution for surface-attached polystyrene beads and DNA tethered magnetic beads, and highlights the influence of mechanical stability for such assay. We also present data where we are able to detect 0.3 nm steps along the z-axis using DNA tethered magnetic beads. Because the data presented here are in agreement with the best resolution obtained with magnetic tweezers, they provide a useful benchmark comparison for setup adjustment and optimization. Specifications table•The data reported here provide a benchmark of the best achievable spatiotemporal resolution of magnetic tweezers for a surface-attached reference bead and a DNA tethered magnetic bead.•The data will be useful to evaluate the resolution of magnetic tweezers, and other camera-based detection assays, such as acoustic force spectroscopy and tethered particle motion.•The data identifies several sources of noise that deteriorate the spatiotemporal resolution of magnetic tweezers.1ADτ over all intervals of length ADτADτ until it increases at longer ADτ, indicating that the drift is now dominating the noise at longer integration time. Therefore, the AD of traces from experiment presenting a large drift  a surface-attached polystyrene reference bead at 50x and 100x magnifications and several acquisition frequencies, subjected to various drift in order to characterize the limiting factors for the best achievable resolution in our assay  and 3; tgctg, 5′-BIO-cacgctcaatctgacaggtg, and the Phusion High-Fidelity DNA Polymerase, following the manufacturers’ instructions . The amplified fragment was purified using the MonarchTM PCR Purification Kit .5Flow cells were assembled from two glass coverslips  separated by a double-layer of Parafilm  carved with a scalpel to form a channel. Inlet and outlet holes were drilled on the top coverslip with a sandblaster . Both coverslips were washed by sonication in a 2% (V/V) Hellmanex III  aqueous solution for 15 min, thoroughly rinsed with deionized water and dried at ∼80 °C. Preceding flow cell assembly, the top surface of the bottom coverslip was spread with 4 µl of 3 µm polystyrene bead solution, made from a 1:150 dilution of the stock  in deionized water further diluted 10-times in ethanol. The coverslip was heated at ∼150 °C for 3 min on a hot plate to ensure fixation of the polystyrene beads to served as reference beads during MT experiments. To finally assemble the flow cell, the carved Parafilm was sandwiched between the two coverslips, such that the two holes of the top coverslips were aligned with the channel, and melted at ∼90 °C for ∼30 s.6After mounting the flow cell on the microscope, 3 mL of PBS buffer were flushed through the flow cell. The flow cell was left to equilibrate for a few hours after which measurements on melted reference beads were performed.For measurements using tethered magnetic beads, anti-digoxigenin  at 25 µg/mL in PBS buffer was first incubated in the flow channel for 15 min. The flow cell was rinsed the 1 mL PBS, followed by a 5 min incubation deionized water and then in PBS buffer complemented with 600 mM NaCl, and rinsed again with 1 mL PBS buffer. Next, BSA  at 1 mg/mL in PBS was incubated for 30 min. During BSA incubation, 20 µL of M270 streptavidin-coated superparamagnetic Dynabeads  were washed twice in the rinsing buffer , mixed with 20 µL of DNA at ∼15 pM in rinsing buffer and incubated for a few minutes. The nucleic acids bound to the magnetic beads were flushed in the flow channel and incubated for 5–10 min to ensure attachment of the DNA construct to anti-digoxigenin. Finally, the excess of magnetic beads was removed by flushing copious amounts of rinsing buffer. Non-specifically and weakly attached magnetic beads were detached from the flow channel bottom surface by applying a force of 15 pN while rotating the magnets back and forth and gently tapping the outlet tube and removed by flushing using rinsing buffer.7All relevant acquisition parameters are specified in the Supplementary Table 1. They include: acquisition speed, objective magnification, shutter time, applied force, number of beads, number of beads suitable for analysis, type of beads (tethered or melted) and DNA construct.The data were analyzed using Python 3.6. Overlapping Allan deviation was calculated using the AllanTools 2019.9 package.