The present invention relates to a method for simultaneously detecting a plurality of pathogens from biologically-derived samples, and a kit for carrying out the method. Specifically, the present invention relates to a method for simultaneously detecting a plurality of pathogens that cause infectious uveitis, one of eye infections from samples such as anterior chamber fluid or vitreous by polymerase chain reaction (PCR), and a kit for carrying out the method.

Sensitive and specific detection of nucleic acids can be achieved using a chemical ligation-based template assisted rapid assay (TARA-L) with simple chemical reactions between probes and without the need for enzymes. Probes are designed to form a ligation product when they anneal to adjacent portions of a target nucleic acid. The ligation products can be detected, such as in immunochromatographic assays. The methods allow for the fast, efficient analysis of biological samples for the presence of nucleic acids and can be used, for example, in point of care settings.

Methods of detecting immunogenic endogenous retroviruses and treating cancer are disclosed herein. In some embodiments, the methods include detecting increased expression of endogenous retroviruses in a tumor sample.

Provided herein are devices, systems, and methods for specimen preparation by employing a combination of capillary and centrifugal forces, along with the addition of reagents at specified steps, followed by on-device sample analysis. For example, provided herein are devices, and methods of use thereof, that collect a sample by capillary force, separate components of the collected sample by centrifugal force, isolate one or more of the separated components by a second application of capillary force, mix the separated components with a first reagent from a storage compartment under centrifugal force, and continue to advance the materials through the device by alternating capillary and centrifugal forces, optionally with the addition of additional reagents from additional storage compartments, until final materials reach a test zone of the device for analysis.

Described herein are uses of fluorescent enveloped virus particles as standards in nanoscale flow cytometry applications. The virus particles comprise a fluorescent dye or a fluorescent protein. A standard ladder comprising a plurality of fluorescent enveloped virus particles of different sizes is also provided. The standards may also comprise marker(s) of interest, and may be used as controls for detection of other viruses or extracellular vesicle, e.g. in diagnostic applications. Methods of producing controls for such applications are provided, including those having desired profiles. The controls may be used for enumeration of markers on microparticles (e.g. extracellular vesicles or viruses). Also described is a modified gammaretrovirus comprising a mutation that abolishes expression of the viral glyco-Gag protein, and having a fluorescent protein inserted in-frame into the proline-rich region (PRR) of the viral env protein. The gammaretrovirus may be M-MLV bearing a mutation that abolishes expression of the glyco-Gal protein, gPr80.

The present invention refers to a method for preparing a linear PCR product from genomic DNA derived from cells of a host subject infected with an retrovirus or a subject suffering from a disease associated with said retrovirus,

wherein the PCR product contains a target sequence comprising an integration site of the retrovirus in the host genomic DNA of the cells, said integration site comprising at least the terminal end of 3-LTR or 5-LTR sequence of the retrovirus and the adjacent host genomic DNA sequence,

wherein the PCR product comprises a first terminus and a second terminus and sequences in the following order:
sequences specific for the first terminus, a sequence comprising at least 6 consecutive random nucleotides followed by a linker sequence, host genomic DNA sequence, at least the terminal end of 3-LTR or 5-LTR sequence of the retrovirus, sequences specific for the second terminus;

wherein the PCR product is prepared by specific steps.

The present invention also refers to a method for determining and longitudinally monitor the dominant leukemic T lymphocyte clone in subjects suffering from Adult T-cell leukemia/lymphoma (ATL),

wherein a linear PCR product is prepared by the method according to the first aspect of the present invention, said PCR product is subjected to multiplex sequencing thereby determining all insertion sites and all shearing sites, the shearing sites are correlated to the respective insertion site,

followed by counting the number of different shear sites for each insertion site representing a specific T lymphocyte clone, removing any PCR duplicate from consideration by eliminating reads that have the same insertion site and the same random tag, and determining the abundance of each specific T lymphocyte clone therefrom.

The present invention refers to a method for preparing a linear PCR product from genomic DNA derived from cells of a host subject infected with an retrovirus or a subject suffering from a disease associated with said retrovirus,

wherein the PCR product contains a target sequence comprising an integration site of the retrovirus in the host genomic DNA of the cells, said integration site comprising at least the terminal end of 3-LTR or 5-LTR sequence of the retrovirus and the adjacent host genomic DNA sequence,

wherein the PCR product comprises a first terminus and a second terminus and sequences in the following order:
sequences specific for the first terminus, a sequence comprising at least 6 consecutive random nucleotides followed by a linker sequence, host genomic DNA sequence, at least the terminal end of 3-LTR or 5-LTR sequence of the retrovirus, sequences specific for the second terminus;

wherein the PCR product is prepared by specific steps.

The present invention also refers to a method for determining and longitudinally monitor the dominant leukemic T lymphocyte clone in subjects suffering from Adult T-cell leukemia/lymphoma (ATL),

wherein a linear PCR product is prepared by the method according to the first aspect of the present invention, said PCR product is subjected to multiplex sequencing thereby determining all insertion sites and all shearing sites, the shearing sites are correlated to the respective insertion site,

followed by counting the number of different shear sites for each insertion site representing a specific T lymphocyte clone, removing any PCR duplicate from consideration by eliminating reads that have the same insertion site and the same random tag, and determining the abundance of each specific T lymphocyte clone therefrom.

The present disclosure relates to real-time quantification using loop-mediated isothermal amplification, in particular real-time colorimetric reverse transcription quantitative loop-mediated isothermal amplification (RT-qLAMP). In some embodiments, RT-qLAMP is used to diagnose the presence of and also quantitate the amount of Zika virus in a sample.

Provided are methods of detecting replication competent retrovirus in a sample containing a cell transduced with a viral vector particle encoding a recombinant and/or heterologous molecule, e.g., heterologous gene product. The methods may include assessing transcription of one or more target genes, such as viral genes, that are expressed in a retrovirus but not expressed in the viral vector particle. Replication competent retrovirus may be determined to be present if the levels of RNA of the one or more target genes is higher than a reference value, which can be measured directly or indirectly, including from a positive control sample containing RNA from the respective target gene at a known level and/or at or above the limit of detection of the assay.

Herein disclosed are rapid real-time isothermal multiplex methods of detecting, identifying and quantifying bacterial, viral, and protozoan nucleic acids in a sample. These include contacting the sample with two or more sets of pathogen-specific reverse transcription loop-mediated isothermal amplification primers and novel oligofluorophores specific for the target bacterial, viral, and parasitic nucleic acids of interest such as human immunodeficiency virus, Ebola virus, Marburg virus, Yellow fever virus, hepatitis-B virus, Lassa fever virus, Plasmodium, hepatitis-C virus, hepatitis-E virus, dengue virus, Chikungunya virus, Japanese Encephalitis virus, Middle Eastern Respiratory Syndrome Corona virus, Mycobacterium, West Nile virus, Cytomegalovirus, Parvovirus, Leishmania, Trypanosoma, and Zika virus nucleic acids, under conditions sufficient to produce detectable real-time amplification signals in about 10 to 40 minutes. The amplification signals are produced by pathogen-specific fluorogenic labels included in one or more of the primers. Also, novel reaction and sample lysis buffers, primers, and kits for rapid multiplex detection, quantification, and identification of bacterial, viral, and protozoan nucleic acids by real-time isothermal amplification are herein disclosed.