FIELD OF INVENTION

The present invention relates to a method for modulating the accessibility of host nucleic acids (HNA) in an acellular fraction of a biological fluid. The present invention further relates to a method for detecting infectious agents in an acellular fraction of a biological fluid.

BACKGROUND OF INVENTION

The detection of pathogenic infectious agents in biological fluids should be performed in the shortest possible time, in particular in the case of septicemia, for which the mortality remains high in spite of the broad range of antibiotics which are available to doctors.

The level of infectious agents in a patient's body fluid is generally very low compared to host components from the subject. Typical biological samples like plasma, serum, cerebrospinal fluid devoid of living or dead cells discarded by centrifugation still contains high amounts of cell free HNA. In a particular case, circulating free cells (not associated with other cells), such as host cells, or fetal cells in a case of pregnancy, can release HNA.

Methods to decrease the amount of host nucleic acids are described in WO2010/062354 and WO2014/114896 and consist in treating biological fluids containing cells with detergent for disrupting the cellular membranes and then treating the resulting sample with nucleases (enzymes having DNase and/or RNase activity). However, these approaches have been of partial efficacy as it remains HNA that is resistant to nucleases in the treated sample.

The Applicant surprisingly demonstrates here that incubation of an acellular fraction of a biological fluid with detergent and nucleases improves the detection of infectious agents in a sample. As a result, the Applicant provides a new method for strongly decreasing the concentration of HNA in an acellular fraction of a biological sample, which results in a better detection of infectious agents in said sample.

SUMMARY

The present invention relates to an in vitro method for isolating, amplifying, and sequencing infectious agents' nucleic acids from an acellular fraction of a biological fluid.

In one embodiment, the method comprises contacting the acellular fraction of a biological fluid with at least one detergent and at least one nucleic acids-digesting enzyme.

In one embodiment, the method further comprises the extraction and sequencing of the infectious agents' nucleic acids.

In one embodiment, the method according to the invention comprises the steps of:a) contacting the acellular fraction of a biological fluid with at least one detergent and at least one nucleic acids-digesting enzyme,b) extracting infectious agents' nucleic acids,c) optionally, reverse-transcribing infectious agents' genomic RNAs,d) amplifying infectious agents' genomic DNA and/or infectious agents' cDNA, ande) sequencing amplified infectious agents' genomic DNA and/or infectious agents' cDNA.

In one embodiment, the sequencing of the infectious agents' nucleic acids is carried out by Next Generation Sequencing (NGS).

In one embodiment, the infectious agent is selected from the group comprising viruses, bacteria, fungi, protozoans, and parasites.

In one embodiment, the at least one detergent is selected from the group comprising non-ionic detergent, zwitterionic detergent, and anionic detergent.

In one embodiment, the at least one detergent is a non-ionic detergent.

In one embodiment, the at least one detergent is selected from the group comprising saponin, Triton, NP-40, Tween and derivative detergents thereof.

In one embodiment, the at least one detergent is saponin.

In one embodiment, the at least one detergent is sapogenin with a sapogenin content of about 5% to about 50%.

In one embodiment, the at least one nucleic acids-digesting enzyme is a nuclease having DNase and/or RNase activity.

In one embodiment, the at least one nucleic acids-digesting enzyme is selected from the group comprising baseline-zero DNase, benzonase, DNase I, DNase II, endonuclease III, endonuclease IV, endonuclease V, endonuclease VIII, exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, exonuclease T, lambda exonuclease, micrococcal nuclease, mung bean nuclease, nuclease BAL-31, nuclease P1, nuclease S1, T4 endonuclease V, T5 exonuclease, T7 endonuclease I, and T7 exonuclease.

In one embodiment, the presence of residual cells within the fraction does not affect the accessibility or the enrichment of the infectious agent in the fraction.

The present invention also relates to a kit-of-parts.

In one embodiment, the kit-of-parts for carrying out the methods according to the present invention.

In one embodiment, the kit-of-parts comprises:a) at least one detergent,b) at least one nucleic acids-digesting enzyme.

In one embodiment, the kit-of-parts comprises:a) at least one detergent, selected from the group comprising saponin, Triton, NP-40, Tween or a derivative thereof,b) at least one nucleic acids-digesting enzyme, selected from the group comprising baseline-zero DNase, benzonase, DNase I, DNase II, endonuclease III, endonuclease IV, endonuclease V, endonuclease VIII, exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, exonuclease T, lambda exonuclease, micrococcal nuclease, mung bean nuclease, nuclease BAL-31, nuclease P1, nuclease S1, T4 endonuclease V, T5 exonuclease, T7 endonuclease I, and T7 exonuclease.

In one embodiment, the kit-of-parts may comprise reagents. In one embodiment, the kit-of-parts may comprise instructions for use in a method according to the present invention.

Thus, in one embodiment, the kit-of-parts comprises:a) at least one detergent, selected from the group comprising saponin, Triton, NP-40, Tween or a derivative thereof,b) at least one nucleic acids-digesting enzyme, selected from the group comprising baseline-zero DNase, benzonase, DNase I, DNase II, endonuclease III, endonuclease IV, endonuclease V, endonuclease VIII, exonuclease I, exonuclease III, exonuclease V, exonuclease VII, exonuclease VIII, exonuclease T, lambda exonuclease, micrococcal nuclease, mung bean nuclease, nuclease BAL-31, nuclease P1, nuclease S1, T4 endonuclease V, T5 exonuclease, T7 endonuclease I, and T7 exonuclease,c) optionally, reagents, andd) optionally, instructions for use in a method according to the present invention.

In one embodiment, the reagents are selected from the group comprising nucleic acid extraction reagents, reverse transcription reagents, nucleic acid amplification reagents, nucleic acid clean-up reagents, nucleic acid size-selection reagents, nucleic acid quantification reagents, nucleic acid library preparation reagents, nucleic acid library sequencing reagents, and internal controls.

In one embodiment, the reagents comprise nucleic acid extraction reagents, reverse transcriptase, vent exo-DNA polymerase, dNTPs, oligonucleotide primers with sequence set forth as any of SEQ ID NO: 1 or 3-13, oligonucleotide primers with sequence set forth as SEQ ID NO: 2, exonuclease I and/or SPRI beads.

Definitions

In the present invention, the following terms have the following meanings:“Acellular fraction” refers to a biological fluid depleted in about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, preferably 99%, more preferably 100% of its cellular content. The acellular fraction of a biological fluid may be obtained by any well-known technique in the art. For example, in one embodiment where the biological fluid is blood, said acellular fraction may be obtained from a centrifugation step from about 100 to about 500 g for about 5 minutes to about 20 minutes, preferably at 300 g for 10 minutes.“Host nucleic acids” or “HNA” refer to nucleic acids issued from a subject.“Modulating” refers to an improvement or increase in the accessibility of HNA.“Primer” refers to an oligonucleotide that is capable of hybridizing or annealing with a nucleic acid and serving as an initiation site for nucleotide polymerization under appropriate conditions, such as the presence of nucleoside triphosphates and an enzyme for polymerization, such as DNA or RNA polymerase or reverse transcriptase, in an appropriate buffer and at a suitable temperature.“Amplification” refers to the process of producing multiple copies, i.e., at least 2 copies, of a desired template sequence. Techniques to amplify nucleic acids are well known to the skilled artisan, and include specific amplification methods as well as random amplification methods.“Reverse transcription” refers to the replication of RNA using a RNA-directed DNA polymerase (reverse transcriptase, RT) to produce complementary strands of DNA (cDNA). The reverse-transcription of RNAs may be carried out by techniques well known to the skilled artisan, using a reverse transcriptase enzyme and a mix of 4 deoxyribonucleotides triphosphate (dNTPs), namely deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and (deoxy)thymidine triphosphate (dTTP).“Subject” refers to any species such as for example domestic animals (i.e., farm fish, pets, ruminants, pigs, horses), wide-life animals and human. In one embodiment, a subject may be a “patient”, i.e., a mammal or more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease.“Internal control” refers to one or more materials used in an assay as a point of reference and/or comparison in order to make judgements as to the presence, absence or level of one or more factors being analyzed. Some internal control may comprise negative or positive control samples. Negative controls are samples known to lack one or more factors being analyzed. Positive controls are samples known to comprise one or more factors being analyzed. In one embodiment, the internal control can be an internal control nucleic acid sequence that is added in the sample at a given time point or at given time points, at a known concentration. In one embodiment, the internal control can be a virus such as a bacteriophage that is added in the sample at a given time point or at given time points, at a known concentration.“About” preceding a figure means plus or less 10% of the value of said figure.

