KR20050016987A - Diagnosis of sepsis using mitochondrial nucleic acid assays - Google Patents

Abstract

The present invention provides an analysis method for detecting the sepsis status of a subject car by measuring the relative amount of mitochondrial nucleic acid in the subject body. Analytical methods of the present invention may include PCR assays, wherein such semiquantitative or quantitative PCR amplifies the mitochondrial sequence and a reference sequence such as, for example, a genomic sequence at the same time. The information obtained by such an analysis can be used to determine the ratio of mitochondrial nucleic acid to nucleic acid in the subject cell.

Description

Diagnosis method of sepsis using mitochondrial nucleic acid analysis {DIAGNOSIS OF SEPSIS USING MITOCHONDRIAL NUCLEIC ACID ASSAYS}

The present invention relates to the field of diagnostic or prognostic analysis of sepsis.

Sepsis is a potentially life-threatening, systemic clinical symptom that can occur after infection or trauma (Mesters, RM et al. (1996) Thromb Haemost . 75: 902-907; Wheeler AP and Bernard GR (1999) N Engl J Med . 340: 207-214. If there are no signs of an incidental microbial infection, sepsis is usually due to the release of microbial toxins during severe infections, although the septic response may result from surgery, physical trauma, burns, organ transplantation or other symptoms, including pancreatitis. (Balk RA and Bone RC (1989) Crit Care Clin 5: 1-8; Ayres SM (1985) Crit Care Med 13: 864-66). In humans, release of endotoxin derived from the outer membrane of lipopolysaccharide of virtually all Gram-negative bacteria is believed to be a common cause of sepsis.

The sequelae of sepsis is characterized by severe hypotension, multiple organ failure or disorder, and necrotic cell death and is not only the most common cause of intensive mortality, but also severe sepsis, sepsis-induced hypotension, or sepsis (Parrillo, JE et al. (1990) Ann Intern Med 113: 227-42; Manship, L. et al., (1984) Am Surg 50: 94-101; Niederman MS and Fein AM (1990) Clin Chest Med ) 11: 663-65). Despite the development of medical and first aid protocols, mortality in patients with septic shock has been estimated to range from 30% to 50%, depending on the presence of other medical complications (Natanson, C. et al. (1998) Crit. Care Med. 26: 1927-1931; Zeni, F. et al. (1997) Crit Care Med . 25: 1095-1100).

The timing of the treatment protocol for sepsis may be critical to the success of the treatment outcome. Delayed initial treatment can have serious consequences for patients. In addition, the optimal window of therapeutic administration may depend on the stage of development of sepsis. Therefore, rapid and reliable diagnosis of sepsis is the key to efficient treatment.

Sepsis diagnosis technology of the present invention is an acidosis or positive blood test of microorganisms; Testing for changes in leukocyte or platelet counts; Or identification of physical symptoms such as fever, chills, colic, increased respiratory rate, increased heart rate, confusion or delirium (von Landenberg, P and Shoenfeld, Y (2001) IMAJ 3: 439-442; Marshall, JC et al. (1995) Crit Care Med 23 (10): 1638-1652; Vincen, JL et al. (1996) Intensive Care Med 22: 707-710; Le Gall, JR et al. (1996) JAMA 276: 802-810; Knaus, WA et al. (1985) Crit Care Med 13: 818-829. However, the results of such diagnostic techniques may be due to the fact that blood culture results are too late to suspend sepsis successfully, or the sensitivity is insufficient, and the physical symptoms of sepsis may be mistaken for other symptoms, resulting in improper treatment. The value is limited.

Thus, identification of sepsis patients early in the development of sepsis, usually before progressing to septic shock, can be used to monitor the status of patients treated with sepsis or to recognize patients at risk of developing sepsis. Development of diagnostic tests is desired. The development of such diagnostics would also be useful in facilitating the detection of sepsis and the development of new therapeutics.

Summary of the Invention

In many aspects, the present invention provides a method for detecting symptoms of sepsis patients based on the finding that mitochondrial nucleic acids (such as mitochondrial DNA or RNA such as mitochondrial mRNA) are generally lacking in patients with sepsis. In some patients, this method was largely effective in screening sepsis regardless of the cause of sepsis.

In one aspect, the present invention provides a method of diagnosing sepsis disease in a subject in need thereof. The method comprises measuring the relative amount of mitochondrial nucleic acid in a sample obtained from the subject, wherein the measured relative amount of mitochondrial nucleic acid can be an indicator of the subject's sepsis disease state. In another aspect, the present invention provides a method of predicting such risks in subjects in need of predicting the risk of sepsis disease states. The method comprises measuring the relative amount of mitochondrial nucleic acid in a sample obtained from the subject, wherein the measured relative amount of mitochondrial nucleic acid can be an indicator of the risk of sepsis disease status in the subject. In any of these aspects, the subject may suffer from sepsis.

In another aspect, the invention provides a method of monitoring the progress of a sepsis disease state in a subject with sepsis. The method comprises measuring the relative amount of mitochondrial nucleic acid in a sample obtained from the subject at a first time point and a second time point, wherein the difference in the relative amount of mitochondrial nucleic acid measured at the first time point and the second time point is determined by the subject. It can be an indicator of the progress of the sepsis disease state. The method may also include measuring the relative amount of mitochondrial nucleic acid in the sample obtained from the subject at several subsequent time points, or the guess of sepsis in the subject may include predicting or predicting clinical course.

In another aspect, the present invention also provides a method for measuring the efficiency of sepsis treatment. The method comprises measuring the relative amount of mitochondrial nucleic acid in a sample obtained from the test subject (the subject receiving the treatment) and the control subject, wherein the measured phase of the mitochondrial nucleic acid in the sample obtained from the test subject and the control subject, respectively. Differences between masses can be indicative of the therapeutic efficacy.

In another aspect, the present invention provides a diagnostic kit for use in determining the severity of sepsis disease state in a subject by measuring the relative amount of mitochondrial nucleic acid in a sample obtained from the subject. The kit may contain mitochondrial nucleic acid primers and nuclear nucleic acid primers.

In another embodiment, the subject may be a human (eg, newborn, or elderly, or immunocompromised) or non-human animal (such as an animal model of sepsis, such as LPS-induced sepsis). . In some embodiments, the subject can be diagnosed with sepsis or tested for risk of sepsis, or can initiate sepsis treatment for the subject if the relative amount of mitochondrial nucleic acid drops below a certain level. The predetermined level may be arbitrarily expressed as a ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA), for example, when the ratio of standard mtDNA / nDNA is set to 1, in which case the predetermined level may be 0.45 or less. have.

In another embodiment, the sepsis disease state is Gram negative bacterial infection, Gram positive bacterial infection, fungal infection, viral infection, physical trauma, pancreatitis, organ transplant, bleeding, adult respiratory distress syndrome, burn, trauma, chemotherapy or ionization It may be due to exposure to light. The sample can be, for example, a peripheral blood sample.

In another embodiment, the relative amount of mitochondrial nucleic acid can guide the severity of sepsis or the success of treatment of sepsis. Mitochondrial nucleic acids can be measured in relative amounts to the amount of nuclear nucleic acid in a subject cell, for example, by using a polymerase chain reaction, such as a quantitative polymerase chain reaction. Here, amplification of the mitochondrial nucleic acid can be compared with amplification of the reference nucleic acid. The polymerase chain reaction can be a real time polymerase chain reaction, where the amplification product can be detected using a hybridization probe.

1 is a standard curve of nuclear gene (ASPOLγ) and mitochondrial gene (CCOI).

As used herein, the term "sepsis" includes "sepsis", "bacteremia", "septicemia", "septic syndrome", and "septic syndrome". septic shock "," severe sepsis ", and" Systemic Inflammatory Response Syndrome "(SIRS), which generally includes preinfective cytokines, adhesion molecules, vasodilators, and reactive oxygen species. Likewise associated with the release of endogenous mediators of infection into the bloodstream and refer to an acute systemic response involving leukocyte counts, body temperature, heart rate and respiratory fluctuations (Bone, RC et al. (1992) Chest 101: 1644 -55; Paterson, RL and NR Webster (2000) JR Coll. Surg. Edinb. , 45: 178-182 ). Sepsis is generally believed to be triggered by an infection or injury and is directed against infectious microorganisms, including Gram-negative and Gram-positive bacteria, fungi, protozoa, and viruses; Or nonspecific host response to an infectious agent due to infection or injury (Bone, RC et al. (1992) Chest 101: 1644-55; Paterson, RL and NR Webster (2000) JR Coll. Surg. Edinb. , 45: 178 -182).

In some embodiments, SIRS can be diagnosed in a patient with two or more of the following symptoms: when the body temperature is above 38 ° C. or below 36 ° C .; Tachycardia with more than 90 beats per minute (greater than 90 beats / minute); More than 20 breaths per minute or PaCO ₂ is less than 4.3 kPa; And if the leukocyte count is greater than 12 x 10 ⁹ / l or less than 4 x 10 ⁹ / l or the immature (band) form is greater than 10%. In another embodiment, sepsis can be defined as SIRS by infection, and severe sepsis can be defined as sepsis with evidence of long-term hypoperfusion. In another embodiment, septic shock can be defined as severe sepsis with hypotension (heart contraction BP <90 mmHG) despite the need for vasoconstriction / muscular contraction for proper conscious recovery or blood pressure maintenance (Paterson, RL). And NR Webster (2000) JR Coll. Surg. Edinb. , 45: 178-182 ). If sepsis is not diagnosed and treated in time, it can develop into septic shock, which can cause life-threatening lowering of blood pressure, and the inflammation-related effects of sepsis damage tissues, leading to progressive organ failure and organ failure. Can lead.

Sepsis can be caused by kidneys (such as due to upper ureter infection); liver; Gallbladder; Intestines (such as peritonitis); Skin (eg, cellulitis); Lung (such as bacterial pneumonia); Urogenital tract (such as urinary sepsis); Or due to local infection of organs such as the brain and spinal cord (eg bacterial meningitis, meningitis) or due to systemic symptoms such as toxic shock syndrome. Sepsis also includes chemotherapy, acute pancreatitis, surgical procedures, physical trauma, burns, organ transplantation, multiple organ dysfunction syndromes (MODS), acute respiratory distress syndrome (ARDS), and acute lung injury ( May occur as a result of non-infectious attacks such as ALI: Acute Lung Injury, Disseminated Intravascular Coagulation (DIC), bleeding, or exposure to ionizing rays (Bone, RC et al. (1992) Chest 101 : 1644-55; Paterson, RL and NR Webster (2000) JR Coll. Surg. Edinb. , 45: 178-182 ).

The physiological symptoms of sepsis include, from diarrhea for mucus, chills, fever, weakness, nausea, vomiting, and diarrhea for severe hypotension, a decrease in mental alertness, and confusion, kidneys causing continuous multiple organ failure or disorders (eg, hypoxia) Dysfunction or disorder; dysfunction or disorder of the lung function leading to dyspnea and hypoxic concentrations in the blood; dysfunction or disorder of the heart function causing blood retention and swelling), and necrotic, which may be characteristic symptoms of septic shock Apoptotic cell death occurs continuously.

In another embodiment, the methods of the present invention further comprise mitochondrial DNA (mt DNA) or mitochondrial RNA in a sample, such as a subject's peripheral blood sample or cell fraction thereof, to determine whether mitochondrial nucleic acid levels are indicative of signs of sepsis. And methods for quantifying mitochondrial nucleic acids such as, for example, mRNA (mt mRNA). "Sample" means any biological fluid, cell, or peripheral blood, lymphocytes (such as B cells, CD4 T cells, CD8 T cells), saliva, urine, wounds, catheter injection sites from a subject, or cell lines derived therefrom. It can be an organization and there are no special restrictions. In another aspect of the invention, samples used in the assays of the invention may be obtained by necropsy or biopsy from various tissues, such as, for example, the heart, brain, lung, kidney, fat, spleen or liver, or cells derived therefrom. Can be.

In another embodiment, the methods of the present invention also include assays for determining the relative amounts of mitochondrial nucleic acid in a subject, such as a subject deemed suffering from sepsis or deemed at risk of sepsis. The subject may be, for example, a human patient being treated for an acute infection or may be a member of a group vulnerable to sepsis. In some embodiments, the subject may be a non-human animal, such as a pet or domestic animal such as a dog, pig, sheep, cow, chicken or turkey. In some embodiments, the subject may be an animal model of sepsis as known to or skilled in the art, and may be a rat, mouse, sheep, pig, baboon monkey, rhesus monkey or dog.

Assays of the present invention can include PCR analysis, such as RT PCR or semi-quantitative or quantitative PCR, which involves co-amplifying a mitochondrial sequence with a reference sequence, such as a genomic sequence. Assays of the invention may also include hybridization assays, such as RNA or DNA hybridization assays, using DNA or RNA samples of mitochondria and nuclei and mitochondria and reference (eg, genome or cDNA) sequences as probes. . The information obtained from this analysis can be evaluated to determine the ratio of mitochondrial nucleic acid to nuclear nucleic acid (such as mt DNA to n DNA or mt mRNA to nuclear RNA (nRNA)) in the subject's cells or tissues.

In various embodiments of the present invention, these assays can thus provide clinical information before sepsis is severe or severe enough to reach septic shock. Deficiency of mitochondrial nucleic acids (such as mt DNA or mt RNA) can be reversed by administering an appropriate therapeutic agent, eg, by administering an appropriately broad spectrum of antibiotics. Severe symptoms of sepsis, including septic shock, have mitochondrial nucleic acid (such as mt DNA or mt RNA) levels of approximately 50%, 40%, 30%, 20%, or 10 of normal when measured with reference to a control sample or a known standard. Can occur when lowered to less than%. Interference in clinical progression of sepsis also suggests that mitochondrial nucleic acid to nuclear nucleic acid ratios (such as mtDNA to nDNA ratios or mt mRNA to nRNA ratios) can be measured with 0.5, 0.45, 0.4, 0.35, or Likewise, it can be done when lowered below the threshold.

In another embodiment, diagnostic information may be provided by measuring the rate of change in relative concentrations of mitochondrial nucleic acid (such as mtDNA or mtRNA) over time. In some subjects, a relatively rapid decrease in the relative amounts of mitochondrial nucleic acid (such as mtDNA or mtRNA) can be an indication of sepsis. For periods within 8 to 10 days, the relative amount of mitochondrial nucleic acid (eg mt DNA for nDNA or mt mRNA for nDNA) relative to nuclear nucleic acid is relatively high to approximately 50% (or 40% in some cases). Rapid decline may indicate an onset of sepsis in the patient, and thus indicate that more or less intensive observation is required and that treatment with antibiotics or other antiseptic therapies is needed.

In another embodiment, the present invention also does not need to measure the mtDNA copy number itself, for example, thereby determining the relative amount of mitochondrial nucleic acid (such as mtDNA or mt mRNA) relative to nuclear nucleic acid (such as nDNA or nRNA). It also provides a protocol that makes it easy to do so. In some aspects, this approach can simplify the diagnostic assay of the present invention. For example, as shown in FIG. 1, numbers representing nuclear genome-equivalent (30 to 30,000) are assigned to nDNA amplification standards, as measured by extrapolating control human DNA of known nuclear-genomic-equivalent concentrations. It is assigned. These same numbers are randomly assigned to the corresponding standard curve for the mitochondrial gene (although they do not represent the calculated copy number of the mitochondrial gene). In another approach, a number representing a nuclear-genomic-equivalent can be randomly assigned to an nDNA amplification standard, e.g. based solely on sample dilution (so that the number represents a relative copy number rather than the absolute value of the nuclear-genomic equivalent). These random numbers can be similarly assigned to the mtDNA amplification standard. The analytical results of the present invention can be expressed, for example, as the ratio of mtDNA to nDNA without having to measure absolute mtDNA copy number. In such embodiments, it may be desirable to use an initial concentration of sample DNA or RNA, which provides sufficient PCR templates such that the number of amplification cycles is the most reliable result, such as at least 5-15 and up to 15-40. .

The present invention thus provides a method for comparing the relative abundance of nucleic acid sequences, which comprises the following steps:

a) measuring amplification momentum of nuclear DNA or RNA sequences under nuclear amplification reaction conditions of the first nuclear control sample and the second nuclear control sample to obtain control nuclear amplification measurements (where the nuclear DNA of the first and second nuclear control samples or RNA sequence concentrations are different);

b) construct a control nuclear DNA or RNA sequence dataset from control nuclear amplification measurements to obtain a model standard relationship between amplification momentum and concentration for nuclear DNA or RNA sequences;

c) measuring the amplification momentum of the mitochondrial DNA or RNA sequence under the mitochondrial amplification reaction conditions of the first mitochondrial control sample and the second mitochondrial control sample to obtain a control mitochondrial amplification measurement (where mitochondrial DNA of the first and second mitochondrial control samples) Or RNA sequence concentrations are different);

d) construct a control mitochondrial DNA or RNA sequence dataset from control mitochondrial amplification measurements to obtain model standard relationships between amplification momentum and concentration for mitochondrial DNA or RNA sequences;

e) measuring the amplification momentum of nuclear DNA or RNA sequences in the test sample under nuclear amplification reaction conditions to obtain test sample nuclear amplification measurements;

f) a model standard relationship between amplification momentum and concentration for nuclear DNA or RNA sequences is applied to test sample nuclear amplification measurements to obtain test sample nuclear DNA or RNA sequence concentration measurements;

g) measuring the amplification momentum of mitochondrial DNA or RNA sequences in the test sample under mitochondrial amplification reaction conditions to obtain test sample mitochondrial amplification measurements;

h) model standard relationships between amplification momentum and concentration for mitochondrial DNA or RNA sequences are applied to test sample mitochondrial amplification measurements to obtain test sample mitochondrial DNA or RNA sequence concentration measurements;

i) Compare the test sample nuclear DNA or RNA sequence concentration measurements with the test sample mitochondrial DNA or RNA sequence concentration measurements to determine the relative concentrations of mitochondrial DNA or RNA sequences with respect to the nuclear DNA or RNA sequences in the test sample.

In another embodiment, the methods and kits of the present invention can be used to identify those subjects in a vulnerable group at very high risk of acute infection, and consequently at risk of sepsis. For example, newborns or fetuses and very young children (under 2 years of age) are particularly vulnerable to infections leading to sepsis as much as the elderly and those with immunodeficiency (including those with severe physical trauma, such as burn patients). In some embodiments of the invention, subjects diagnosed as having HIV or cancer, suspected of having HIV or cancer, or at risk are excluded from the methods of the invention so that the present invention is directed to HIV infection or cancer. Methods for determining the relative amounts of mitochondrial nucleic acid in a sample obtained from a patient who is diagnosed as having, suspected of having an HIV infection or cancer, or who is not at risk. In some embodiments, the methods and kits of the invention can be used to identify or monitor sepsis disease status in non-human animals, such as, for example, pets, livestock, or experimental animals.

Current therapies include treating vulnerable subjects with high fever or treating them with lumbar puncture to eliminate meningitis. Thus, under current sepsis treatment plans, many patients who do not have sepsis will also receive unnecessary sepsis treatment, which is undesirable in many ways. For example, the administration of broad-spectrum antibiotics is undesirable due to the risks associated with antibiotic therapy, such as drug allergies, hearing damage, or internal organ damage due to lack of drug, and development of antibiotic-resistant bacteria against bacteria, such as lumbar puncture. The treatment is relatively invasive to the patient and causes more trauma.

By using the rapid and relatively non-invasive method of the present invention, only the patients identified as having sepsis can be treated as necessary. Such patients can be treated with infection-specific therapeutic agents when possible; Can be treated early or more strongly with a broad spectrum of antibiotics; Or by a process such as lumbar puncture. Unlike the sepsis detection method, which depends on the presence of microorganisms in the bloodstream, the method of the present invention can be used to detect sepsis when the subject is treated with antiseptic drugs such as new antibiotics.

In another embodiment, the methods of the present invention are also available for identifying patients with favorable sepsis or subjects susceptible to sepsis if they interfere early. Such identification can help health practitioners to treat earlier and perhaps more aggressive therapies. Thus, patients who have been shown to be severely deficient in relative mitochondrial nucleic acid levels, such as relative mtDNA or mtRNA levels, would be advantageous to receive thorough, persistent and / or various antibiotic treatments. Likewise, it may be desirable to postpone surgery in patients who lack relative mitochondrial nucleic acid levels, such as relative mtDNA or mtRNA levels.

In another aspect, the methods of the present invention can also be used in experimental models of sepsis to test the effectiveness of new therapies for the treatment of sepsis. Animal models of sepsis include: administering bacterial endotoxin (lipopolysaccharide, LPS) to simulate sepsis caused by Gram negative bacteria; Chemotherapy-induced infection; Cecal junction and double perforation in rats for modeling acute respiratory distress syndrome; Or other experimental models capable of simulating sepsis symptoms. Descriptions of animal models of sepsis can be found in the following documents, which are incorporated herein by reference, and are not limited to these: Bhatti AR and Micusan VV (1996) Microbios 86 (349): 247-53; Redl H, et al (1993) Immunobiologyl 87 (3-5): 330-45; Fink MP and Heard SO (1990) J Surg Res. 49 (2): 186-96; Dunn DL (1988) Transplantation 5 (2): 424-9; Bohnen JM, et al. (1988) Can J Microbiol. 34 (3): 323-6; Mela-Riker L, et al. (1988) Circ Shock. 25 (4): 231-44; Walker JF, et al. (1986) Am J Kidney Dis. 8 (2): 88-97; Lopez-Garrido J, et al. (1987) Lab Invest. 56 (5): 534-43; Quimby F and Nguyen HT (1985) Crit Rev Microbiol. 12 (1): 1-44; Noel GJ, et al. (1985) Pediatr Res. 19 (3): 297-9; Moesgaard F, et al (1983) Eur J Clin Microbiol. 2 (5): 459-62; Hinshaw LB, et al. (1976) Surg Gynecol Obstet. 142 (6): 893-900; Tieffenberg J., et al. (1978) Infect Immun. 19 (2): 481-5; Wing D. A., et al. (1978) J Lab Clin Med. 92 (2): 239-51.

In another aspect, the present invention provides a kit having elements for use in the method of the present invention. Such kits may include PCR components, including PCR primers specific for mtDNA or mtRNA sequences and PCR primers specific for nDNA or nRNA sequences, as detailed below. Such kits may also include written instructions for carrying out the methods of the present invention as described herein.

In yet another embodiment, a variety of different techniques can be used to measure the relative amounts of DNA or RNA in mitochondrial cells. Quantitative PCR methods are described, for example, in the following documents, all of which are incorporated herein by reference: US Pat. No. 6,180,349 to Ginzinger et al., Jan. 30, 2001; U.S. Patent No. 6,033,854 to Kurnit et al., March 7, 2000; And US Patent No. 5,972,602 to Hyland et al., October 26, 1999; Song, J. et al. (2001) Diabetes Care 24: 865-869. The DNA or RNA sequence of the mitochondria may be any mitochondrione specific nucleotide sequence, including ATP synthase 6, GenBank Accession No. AF368271; tRNA-Leu, GenBank Accession No. S49541; NADH dehydrogenase subunit 5 (MTND5), GenBank Accession No. AF339085; IDL, GenBank Accession No. AF079515; Cytochrome b, GenBank Accession No. AF254896, CCOI or other suitable mitochondrione specific nucleotide sequences are included, but are not limited to these. The DNA or RNA sequence of the nucleus may be any sequence, and any and all of these may be human 28S rRNA sequences, ASPOL-gamma sequences, beta-globin sequences, or other appropriate nuclear DNA or RNA sequences, and are not particularly limited. Amplification probes are described, for example, in Sambrook et al. (Molecular CLoning; A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989) or Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & SOns, Design according to known methods as described in 1994).

In another aspect, the methods of the present invention may be combined with other therapies, prophylaxis or diagnostic methods of sepsis described in the following enumerated references, which are incorporated herein by reference, and such other methods are limited to those disclosed in these documents. Not: von Landenberg, P. and Shoenfeld, Y (2001) IMAJ 3: 439-442; Marshall, JC, Cook, DJ, Christou, NV, Bernard, GR, Sprung, CL, Sibbald, WJ (1995) Cirt Care Med 23 (10): 1638-1652; Vincent JL, Moreno R., Takala J., Willarts S., (1996) Intensive Care Med 22: 707-710; Le Gall JR, Klar J., Lemeshow S. (1996) JAMA 276: 802-810; Knaus, WA, Draper, E., Wagner, D., and Zimmerman, J. (1985) Crit Care Med 1 3: 818-829; Knaus WA, Wagner DP, Draper EA, Zimmerman JE et al. (1991) Chest 100: 1619-38; BOne, RC, Balk, RA, Cerra, FB, Dellinger, RP, Fein, AM, Knaus, WA Schein, RMH, Sibbald, WJ (1992) Chest 101: 1644-55; US Patent No. 6,303,321 to Tracey et al. October 16, 2001; US Patent No. 5,993,811 to Becker et al., Nov. 30, 1999; US Patent No. 5,830,679 to Bianchi et al. 3 November 1998; US Patent No. 5,804,370 to Romaschin et al., Sep. 8, 1998; US Patent No. 5,780,237 to Bursten et al., July 14, 1998; U.S. Patent No. 5,639,617 to Bohuon, Jun. 17, 1997; US Patent No. 5,545,721 to Carroll et al., Filed Aug. 13, 1996; Or US Pat. No. 5,998,482 to David et al., December 7, 1999. Alternatively or additionally, such patients may be treated with mitochondrial therapeutics that are beneficial components of the mitochondria, such as mitochondrial enzyme cofactors or precursors. In some embodiments, such mitochondrial therapeutics include, for example, riboflavin (vitamin B2), coenzyme Q10, vitamin B1 (thiamine), vitamin B12, vitamin K, 1-acetyl carnitine, N-acetyl cysteine and nicotinamide.

Example 1

Sepsis is associated with a severe decrease in blood cell mtDNA content. Assays are provided for monitoring mitochondrial DNA levels in, for example, subjects with sepsis. The methods of the present invention can be used to assess the efficacy of antiseptic drugs and to diagnose sepsis in individuals suspected of being at risk of sepsis or in patients already suffering from sepsis.

Materials and methods

Longitudinal blood samples may be taken from the subject. Total DNA can be extracted from blood cells and amplified and quantified by both real and mitochondrial genes by real-time PCR using hybridization probes. mtDNA levels can be expressed as the ratio of mitochondrial DNA to nuclear DNA (mtDNA / nDNA).

Sampling and DNA Extraction

Buffycoats were taken from the blood samples and stored frozen at -70 ° C until use. 200 μl of the pale yellow film was extracted using a QIAamp DNA Blood Mini kit (QIAGEN, Missisauga, Ontario, Canada) according to the manufacturer's protocol and resuspended in 200 μl of elution buffer. As a standard curve, similar samples were taken from control volunteers to extract and collect DNA. The nuclear genome equivalent (g.eq) content of the DNA pool is known from the known nuclear g.eq. Concentration kits can be obtained by extrapolation using human DNA (Roche Molecular Bichemicals, Laval, Quebec, Canada).

Quantitative Real-Time PCR

For the mtDNA CCOI gene, the CCOI1F 5'-TTCGCCGACC GTTGACTATT-3 '(SEQ ID NO: 1) and CCOI2R 5'-AAGATTATTACAAATGCATGGGC-3' (SEQ ID NO: 2) primers can be used for PCR amplification, and the oligonucleotide 3 ' -Fluorescein-labeled CCOIPR1 5'-GCCAGCCAGGCAACCTTCTAGG-F-3 '(SEQ ID NO: 3) and 5'LC Red640-labeled CCOIPR2 5'-AACGACCACATCTACAACGTTATCGTCAC-P-3 with 3' end blocked by phosphate molecule '(SEQ ID NO: 4) can be used as the hybridization probe.

For nDNA ASPOL γ (gamma) genes, ASPG3F 5'-GAGCTGTTGACGGAAAGGAG-3 '(SEQ ID NO: 5) and ASPG45 5'-CAGAAGAGAATCCCGGCTAAG-3' (SEQ ID NO: 6) primers can be used for PCR and oligonucleotides 3'-Fluorescein-labeled ASPGRP1 5'-GAGGCGCTGTTAGAGATCTGTCAGAGA-F-3 '(SEQ ID NO: 7) and 5'LC Red640-labeled, 3'-phosphate-blocked ASPGPR2 5'-L-GGCATTTCCTAAGTGGAAGCAAGCA- P-3 '(SEQ ID NO: 8) can be used as the hybridization probe.

Real-time PCR reactions were performed twice for each gene using the LightCycler FastStart DNA Master Hybridization Probes kit (Roche Molecular Biochemicals, Laval, Quebec, Canada). PCR reactions contained 5 mM MgCl ₂ , 0.5 μm each primer, 0.1 μm 3-fluorescein probe, 0.2 μm 5′LC Red 640 probe and 4 μl of DNA extract in 1:10 dilution in elution buffer. can do. PCR amplification may consist of a 10 minute single denaturation / enzyme activation process at 95 ° C. followed by 45 cycles of 0s / 95 ° C., 10s / 60 ° C., 5s / 72 ° C. and 20 ° C./s temperature transfer rates. The gain settings are F1 = 1, F2 = 8 and a single fluorescence can be obtained at the end of each annealing process. Nuclear g. eq. External standard curves of 30, 300, 3000 and 30000 were included in each LightCycler run and the same nuclei g. eq. Values were used for both nuclear genes (ASPOLγ) and mitochondrial genes (CCOI). This data can be analyzed by correlating it with the corresponding standard curve using the second derivative maximum of each amplification reaction. The result of quantitative PCR is the mean nDNA g. It can be expressed as the relative ratio (mtDNA / nDNA) of the mean mtDNA g.eq. to eq., which ratio is arbitrarily approximately from the fact that the same nuclear g.eq. value is available to produce both standard curves. Is set to 1.0.

In some embodiments, the PCR method of the present invention is a LightCycler Fast Star DNA Master Hybridization Probes kit (Roche Molecular BIochemicals, Laval, Quebec, Canada) or otherwise commercially available ABI Taqman Amplification products with hybridization probes in the same manner as described above using technology (e.g., Applied Biosystems, Foster City, California, USA) using an ABI Prism 7700 instrument to detect accumulation of PCR products continuously during the PCR process It may be a real-time polymerase chain reaction to detect the. Other PCR methods and variations of the methods described above are also acceptable, as described, for example, in the following U.S. patent documents incorporated herein by reference: 6,180,349 (Ginzinger et al; Jan. 30, 2001); 6,033,854 (Kuit et al., March 7, 2000); 5,972,602 (Hyland; October 26, 1999); 5,476,774 and 5,219,727 (Wang; December 19, 1999 and June 15, 1993); 6,174,670 (Wittwer et al., January 16, 2001); 6,143,496 (Brown; 7 November 2000); 6,090,556 (Kato; July 18, 2000); 6,063,568 (Gerdes et al., May 16, 2000).

Example 2

LPS-induced sepsis was used in animal models for sepsis screening. Table 1 shows the mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) ratios in lung and liver tissues 6 and 24 hours after LPS administration to mice.

Relative amounts of mitochondrial DNA in mouse tissues 6 and 24 hours after LPS administration Aid group N mtDNA / nDNA ratio (mean ± SD) Control lungs 6 0.29 ± 0.05 LPS-6 hours lungs 6 0.23 ± 0.04 LPS-24 hours lungs 6 0.25 ± 0.06 Control liver 3 2.40 ± 0.99 LPS-24tlrks liver 3 2.50 ± 0.22

The results indicate that the mtDNA / nDNA ratio tends to decrease significantly in lung tissue of animals with LPS-induced sepsis (P = 0.06).

conclusion

While various embodiments of the present invention have been disclosed, various modifications and applications are possible within the scope of the present invention according to general knowledge of those skilled in the art. Such modifications include the substitution of any aspect of the invention with known equivalents in order to achieve the same result in a substantially identical manner. The numerical range includes the numbers that define the range. The expression "comprising" in this specification is an open concept, that is, substantially meaning "including, but not limited to," and "comprises" also has a corresponding meaning. Reference citations in the specification should not be construed as an admission that the references so cited are prior art of the present invention. All publications cited herein, not limited to patents and patent applications, and U.S. Provisional Application No. 60 / 393,368, filed July 5, 2002, which is the basis for priority claims of the present invention, each such publication is specific and individual. It is hereby incorporated by reference herein and incorporated herein in its entirety. The present invention encompasses substantially all of the various embodiments and modifications with reference to the above embodiments and the accompanying drawings.

<110> THE UNIVERSITY OF BRITISH COLUMBIA <120> DIAGNOSIS OF SEPSIS USING MITOCHONDRIAL NUCLEIC ACID ASSAYS <130> KP-CH-046031 <150> US 60 / 393,368 <151> 2002-07-05 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ttcgccgacc gttgactatt 20 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 aagattatta caaatgcatg ggc 23 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <220> <221> misc_feature <222> (22) <223> 3'-Fluorescein-labeled <400> 3 gccagccagg caaccttcta gg 22 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <220> <221> misc_feature <222> (1) <223> 5'-LC Red640-labeled <400> 4 aacgaccaca tctacaacgt tatcgtcac 29 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gagctgttga cggaaaggag 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cagaagagaa tcccggctaa g 21 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <220> <221> misc_feature <222> (27) <223> 3'-Fluorescein-labeled <400> 7 gaggcgctgt tagagatctg tcagaga 27 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <220> <221> misc_feature <222> (25) <223> 3'-Phostphate-blocked <400> 8 ggcatttcct aagtggaagc aagca 25

Claims (30)

And measuring the relative amount of mitochondrial nucleic acid in a sample obtained from the subject, wherein the relative amount of mitochondrial nucleic acid measured is an indicator of the presence or absence of a sepsis disease state in the subject. A method of diagnosing sepsis disease status in a subject. And measuring a relative amount of mitochondrial nucleic acid in a sample obtained from the subject, wherein the relative amount of mitochondrial nucleic acid measured is an indicator of the risk of developing sepsis disease in the subject. A method of predicting the risk of developing sepsis disease in a subject in need of a prediction of risk. The method of claim 1 or 2, wherein the subject has sepsis. Measuring a relative amount of mitochondrial nucleic acid in a sample obtained from the subject at a first point of time and measuring a relative amount of mitochondrial nucleic acid in a sample obtained from the subject at a second point of time, wherein at the first and second points of time A method for monitoring the progression of sepsis quality or condition in a subject with sepsis, wherein the difference in the relative amount of measured mitochondrial nucleic acid is an indicator of the progress of the sepsis disease state in the subject. The method of claim 1, further comprising determining the relative amount of mitochondrial nucleic acid in the sample obtained from the subject at several subsequent time points. The method of claim 1, further comprising predicting or predicting the clinical course of sepsis in the subject. i) determining the relative amount of mitochondrial nucleic acid in a sample obtained from a control subject; ii) measuring the relative amount of mitochondrial nucleic acid in a sample obtained from a subject under treatment for sepsis, Wherein the difference between said measured relative amounts of mitochondrial nucleic acid in a sample obtained from said test subject and a control subject is used as an index of said sepsis therapeutic efficacy. 8. The method of any one of claims 1 to 7, wherein the sepsis vaginal condition is Gram negative bacterial infection, Gram positive bacterial infection, fungal infection, viral infection, protozoa infection, physical trauma, pancreatitis, organ transplantation, bleeding, adult Sex Respiratory Distress Syndrome, Burn, Surgical Procedure, Chemotherapy, and Sepsis Syndrome due to a situation selected from the group consisting of exposure to ionizing light. The method of any one of claims 1 to 8, wherein the relative amount of the mitochondrial nucleic acid indicates the severity of sepsis or whether the treatment of sepsis is successful. The method of claim 1, wherein the mitochondrial nucleic acid is measured relative to the amount of nuclear nucleic acid in a cell of the subject. The method according to any one of claims 1 to 10, wherein said mitochondrial nucleic acid or said nuclear nucleic acid is measured by polymerase chain reaction. The method of claim 11, wherein the polymerase chain reaction is a quantitative polymerase chain reaction, comparing amplification of mitochondrial nucleic acid with amplification of a reference nucleic acid. The method of claim 12, wherein the polymerase chain reaction is a real time polymerase chain reaction that detects an amplification product using a hybridization probe. The method of any one of claims 1 to 13, wherein when the relative amount of mitochondrial nucleic acid falls below a predetermined level, the subject is diagnosed as having sepsis or is assessed as at risk of developing sepsis. 15. The method of claim 14, wherein the predetermined level is expressed as a ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA), wherein the predetermined level is a ratio of 0.45 or less when the standard mtDNA / nDNA ratio is set to 1. . The method of claim 1, further comprising initiating sepsis treatment in the subject when the relative amount of mitochondrial nucleic acid drops below a predetermined level. The method according to claim 16, wherein the predetermined level of mitochondrial DNA is expressed by the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA), which is 0.45 or less when the standard mtDNA / nDNA ratio is set to 1 How is the ratio of. 18. The method of any one of claims 1 to 17, wherein the subject is a human. The method of any one of claims 1-18, wherein the subject is a newborn, an immunocompromised subject, and an elderly subject. The method of claim 1, wherein the subject is a non-human animal. The method of claim 20, wherein the non-human animal is an animal model of sepsis. The method of claim 21, wherein the animal model of sepsis is LPS-induced sepsis. 23. The method of any one of claims 1 to 22, wherein the sample is a peripheral blood sample. 24. The method of any one of claims 1 to 23 wherein the mitochondrial nucleic acid is DNA. The method of claim 1, wherein the mitochondrial nucleic acid is RNA. The method of claim 25, wherein the RNA is mRNA. i) mitochondrial nucleic acid primers, and ii) nuclear nucleic acid primers It is made, including A diagnostic kit for measuring the severity of sepsis disease state of a subject by measuring the relative amount of mitochondrial nucleic acid in a sample obtained from the subject. The kit of claim 27 wherein the mitochondrial nucleic acid is DNA. 29. The kit of claim 27 or 28 wherein the mitochondrial nucleic acid is RNA. The kit of claim 29 wherein the RNA is mRNA.