WO2016180037A1 - Compositions et procédés de détection de pathogènes dans les infections des voies respiratoires - Google Patents

Compositions et procédés de détection de pathogènes dans les infections des voies respiratoires Download PDF

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WO2016180037A1
WO2016180037A1 PCT/CN2016/000245 CN2016000245W WO2016180037A1 WO 2016180037 A1 WO2016180037 A1 WO 2016180037A1 CN 2016000245 W CN2016000245 W CN 2016000245W WO 2016180037 A1 WO2016180037 A1 WO 2016180037A1
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polynucleotide
seq
nucleic acid
acid sequence
primer
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PCT/CN2016/000245
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English (en)
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Guangxin Xiang
Can Wang
Wanli Xing
Jing Cheng
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Capitalbio Corporation
Tsinghua University
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Publication of WO2016180037A1 publication Critical patent/WO2016180037A1/fr
Priority to ZA2017/07258A priority Critical patent/ZA201707258B/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the present disclosure relates to the field of biotechnology, and specifically, compositions and methods for the detection of respiratory tract infection-related pathogenic organisms, for example, using loop-mediated isothermal amplification (LAMP) primers.
  • LAMP loop-mediated isothermal amplification
  • Respiratory tract infections are common and can plague even the healthiest individuals. Common pathogens in respiratory tract infections include Streptococcus pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) , Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Haemophilus influenzae, Legionella pneumophila, Mycobacterium tuberculosis complex, mycoplasma pneumoniae, and Chlamydia pneumoniae. Respiratory tract infections are the most common diseases in the clinics in China.
  • a plurality of polynucleotides comprising one or more of the polynucleotide sets selected from the group consisting of: (a) a first polynucleotide comprising a nucleic acid sequence of at least about 85%homology with SEQ ID NO: 14; a second polynucleotide comprising a nucleic acid sequence of at least about 85%homology with SEQ ID NO: 15; a third polynucleotide of the formula A-linker-B, wherein A comprises a nucleic acid sequence of at least about 85%homology with SEQ ID NO: 170, the linker comprises between about 0 and about 10 nucleotide residues, and B comprises a nucleic acid sequence of at least about 85%homology with SEQ ID NO: 171; a fourth polynucleotide of the formula A-linker-B, wherein A comprises a nucleic acid sequence of at least about 85%homology with SEQ ID NO:
  • the third polynucleotide in polynucleotide set (a) , can comprise the nucleic acid sequences of SEQ ID NO: 170 and SEQ ID NO: 171, and/or the fourth polynucleotide can comprise the nucleic acid sequences of SEQ ID NO: 172 and SEQ ID NO: 173; in polynucleotide set (c) , the third polynucleotide can comprise the nucleic acid sequences of SEQ ID NO: 174 and SEQ ID NO: 175, and/or the fourth polynucleotide can comprise the nucleic acid sequences of SEQ ID NO: 176 and SEQ ID NO: 177; in polynucleotide set (e) , the third polynucleotide can comprise the nucleic acid sequences of SEQ ID NO: 178 and SEQ ID NO: 179, and/or the fourth polynucleotide can comprise the nucleic acid sequences of SEQ ID NO
  • the third polynucleotide in polynucleotide set (a) , can comprise the nucleic acid sequence of SEQ ID NO: 16, and/or the fourth polynucleotide can comprise the nucleic acid sequence of SEQ ID NO: 17; in polynucleotide set (c) , the third polynucleotide can comprise the nucleic acid sequence of SEQ ID NO: 22, and/or the fourth polynucleotide comprise the nucleic acid sequence of SEQ ID NO: 23; in polynucleotide set (e) , the third polynucleotide can comprise the nucleic acid sequence of SEQ ID NO: 28, and/or the fourth polynucleotide can comprise the nucleic acid sequence of SEQ ID NO: 29; in polynucleotide set (g) , the third polynucleotide can comprise the nucleic acid sequence of SEQ ID NO: 34, and/or the fourth polynucleot
  • the molar ratio between the first and second polynucleotides can be about 1: 1, and/or the molar ratio between the third and fourth polynucleotides can be about 1: 1.
  • the molar ratio between the first, second, third, and fourth polynucleotides is about (0.2 -0.3) : (0.2 -0.3) : (0.8 –2.4) : (0.8 –2.4) .
  • the molar amount of the first polynucleotide can be about 0.2 -0.3 ⁇ mol
  • the molar amount of the second polynucleotide can be about 0.2 -0.3 ⁇ mol
  • the molar amount of the third polynucleotide can be about 0.8 –2.4 ⁇ mol
  • the molar amount of the fourth polynucleotide can be about 0.8 –2.4 ⁇ mol.
  • polynucleotide set (a) can further comprise a fifth polynucleotide comprising a nucleic acid sequence of at least about 85%homology with SEQ ID NO: 18, and/or a sixth polynucleotide comprising a nucleic acid sequence of at least about 85%homology with SEQ ID NO: 19;
  • polynucleotide set (c) can further comprise a fifth polynucleotide comprising a nucleic acid sequence of at least about 85%homology with SEQ ID NO: 24, and/or a sixth polynucleotide comprising a nucleic acid sequence of at least about 85%homology with SEQ ID NO: 25;
  • polynucleotide set (e) can further comprise a fifth polynucleotide comprising a nucleic acid sequence of at least about 85%homology with SEQ ID NO: 30, and/or a sixth polynucleotide comprising a nucleic acid sequence of at least about 85%homology
  • the molar ratio between the fifth and sixth polynucleotides can be about 1: 1.
  • the molar ratio between the first, second, third, fourth, fifth, and sixth polynucleotides can be about (0.2 -0.3) : (0.2 -0.3) : (0.8 –2.4) : (0.8 –2.4) : (0.4 –1.0) : (0.4 –1.0) .
  • the molar amount of the first polynucleotide can be about 0.2 -0.3 ⁇ mol
  • the molar amount of the second polynucleotide can be about 0.2 -0.3 ⁇ mol
  • the molar amount of the third polynucleotide can be about 0.8 –2.4 ⁇ mol
  • the molar amount of the fourth polynucleotide can be about 0.8 –2.4 ⁇ mol
  • the molar amount of the fifth polynucleotide can be about 0.4 –1.0 ⁇ mol
  • the molar amount of the sixth polynucleotide can be about 0.4 –1.0 ⁇ mol.
  • polynucleotide set (b) can comprise a first polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 92, a second polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 93, a third polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 94, and/or a fourth polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 95;
  • polynucleotide set (d) can comprise a first polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 98, a second polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 99, a third polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 100, and/or a fourth polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 101;
  • the linker of the third polynucleotide in polynucleotide set (a) , can be about 4 nucleotide residues in length which are optionally GTAG, and/or the linker of the fourth polynucleotide can be about 6 nucleotide residues in length which are optionally AACAAC; in polynucleotide set (c) , the linker of the third polynucleotide can be about 0 nucleotide residue in length, and/or the linker of the fourth polynucleotide can be about 0 nucleotide residue in length; in polynucleotide set (e) , the linker of the third polynucleotide can be about 0 nucleotide residue in length, and/or the linker of the fourth polynucleotide can be about 0 nucleotide residue in length; in polynucleotide set (g) , the linker of the third polynucleotide can
  • the plurality of polynucleotides can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or all 26 of the polynucleotide sets (a) - (z) .
  • the plurality of polynucleotides can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or all 13 of the polynucleotide sets selected from the group consisting of (a) , (c) , (e) , (g) , (i) , (k) , (m) , (o) , (q) , (s) , (u) , (w) , and (y) .
  • the plurality of polynucleotides can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or all 13 of the polynucleotide sets selected from the group consisting of (b) , (d) , (f) , (h) , (j) , (l) , (n) , (p) , (r) , (t) , (v) , (x) , and (z) .
  • the polynucleotides in one or more of the polynucleotide sets can be primers.
  • the primers are loop-mediated isothermal amplification (LAMP) primers.
  • the primers can be specific for at least one pathogen.
  • the at least one pathogen is a bacterium.
  • the bacterium is selected from the group consisting of Streptococcus pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) , Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Haemophilus influenzae, Legionella pneumophila, Mycobacterium tuberculosis complex, mycoplasma pneumoniae, and Chlamydia pneumoniae.
  • MRSA methicillin-resistant Staphylococcus aureus
  • each of the polynucleotide sets can comprise primers for loop-mediated isothermal amplification (LAMP) of a nucleic acid sequence from a pathogen in respiratory tract infection.
  • the plurality of polynucleotides comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the polynucleotide sets, each of which comprises primers for loop-mediated isothermal amplification (LAMP) of a nucleic acid sequence from a different pathogen selected from the group consisting of Streptococcus pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) , Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Haemophilus influenzae, Legionella pneumophila, Mycobacterium tuberculo
  • kits comprising at least one of the plurality of polynucleotides of any of the preceding embodiments, and optionally an instruction for use.
  • each of the polynucleotide sets is individually packaged.
  • one or more of the polynucleotide sets are packaged together.
  • a chip comprising at least one of the plurality of polynucleotides of any of the preceding embodiments, wherein the polynucleotide (s) is or are optionally immobilized on a substrate.
  • the chip comprises at least one polynucleotide set comprising the first, second, third, and fourth polynucleotide in the set.
  • the at least one polynucleotide set further comprises the fifth and sixth polynucleotide in the set.
  • the chip can be for detecting all thirteen, any twelve, any eleven, any ten, any nine, any eight, any seven, any six, any five, any four, any three, any two, or any one of the pathogens selected from the group consisting of: Streptococcus pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Haemophilus influenzae, Legionella pneumophila, Mycobacterium tuberculosis complex, mycoplasma pneumoniae, and Chlamydia pneumonia.
  • the pathogens selected from the group consisting of: Streptococcus pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae
  • the chip can be an isothermal amplification micro-fluidic chip.
  • a system comprising the kit and/or the chip of any of the preceding embodiments.
  • the system further comprises a reagent for detecting the respiratory tract infection pathogen (s) , and/or an instrument, and/or an amplification product data processor.
  • the reagent does not comprise the plurality of polynucleotides of any of the preceding embodiments.
  • the system can be for detecting all thirteen, any twelve, any eleven, any ten, any nine, any eight, any seven, any six, any five, any four, any three, any two, or any one of the pathogens selected from the group consisting of: Streptococcus pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Haemophilus influenzae, Legionella pneumophila, Mycobacterium tuberculosis complex, mycoplasma pneumoniae, and Chlamydia pneumonia.
  • the pathogens selected from the group consisting of: Streptococcus pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae
  • a method of detecting a pathogen in an individual comprising: contacting a biological sample from an individual with the plurality of polynucleotides, the kit, the chip, and/or the system of any of the preceding embodiments, wherein the biological sample contains or is suspected of containing a nucleic acid of a pathogen; performing an isothermal amplification reaction on the biological sample; detecting an amplification product of the nucleic acid of the pathogen in the biological sample, wherein the presence, absence, or amount of the amplification product indicates the presence, absence, or amount of the nucleic acid in the biological sample, thereby detecting the presence, absence, or amount of the pathogen in the individual.
  • the individual is a human.
  • the pathogen can be selected from the group consisting of: Streptococcus pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Haemophilus influenzae, Legionella pneumophila, Mycobacterium tuberculosis complex, mycoplasma pneumoniae, and Chlamydia pneumonia.
  • Figure 1 is a real-time quantitative amplification curve, according to one aspect of the present disclosure.
  • the results were obtained from the PCR reaction in the reaction wells A1-M1 in which one of the 13 LAMP primers was fixed in each well to detect the respiratory tract infection pathogenic microorganisms.
  • the wells N1 to X1 were without primers. Tests were carried out with 6 kinds of pathogenic bacteria DNA, i.e., SP, E. coli, SMA, LP, MP, and CP as template.
  • Figure 2 is a real-time quantitative amplification curve, according to one aspect of the present disclosure.
  • the results were obtained from the PCR reaction in the reaction wells A2-M2 in which one of the 13 LAMP primers was fixed in each well to detect the respiratory tract infection pathogenic microorganisms.
  • the wells N2 to X2 were without primers.
  • Tests were carried out with 7 kinds of pathogenic bacteria DNA, i.e., SAS, MRSA, KP, PA, AB, HI, and MTC as template.
  • Figure 3 is a real-time quantitative amplification curve, according to one aspect of the present disclosure.
  • the results were obtained from the PCR reaction in the reaction wells A3-M3 in which one of the 13 LAMP primers was fixed in each well to detect the respiratory tract infection pathogenic microorganisms.
  • the wells N3 to X3 were without primers. Tests were carried out with DNA from sputum of pneumonia patients as template.
  • ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.
  • description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6.
  • a sample includes one or more samples
  • a primer includes one or more primers.
  • a biological sample or material can be obtained and used, and can refer to any sample or material obtained from a living or viral (or prion) source or other source of macromolecules and biomolecules, and includes any cell type or tissue of a subject from which nucleic acid or protein or other macromolecule can be obtained.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • isolated nucleic acids that are amplified constitute a biological sample.
  • a template for the LAMP or PCR reaction disclosed herein can be comprised in and/or obtained from a biological sample, for example, a sample from a patient or a subject suspected of respiratory tract infection.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants and processed samples derived therefrom.
  • polynucleotide oligonucleotide, “ “nucleic acid” and “nucleic acid molecule” are used interchangeably herein to refer to a polymeric form of nucleotides of any length, and may comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double-and single-stranded deoxyribonucleic acid ( "DNA” ) , as well as triple-, double-and single-stranded ribonucleic acid ( "RNA” ) .
  • DNA triple-, double-and single-stranded deoxyribonucleic acid
  • RNA triple-, double-and single-stranded ribonucleic acid
  • polynucleotide oligonucleotide, " nucleic acid "and “ nucleic acid molecule” include polydeoxyribonucleotides (containing 2-deoxy-D-ribose) , polyribonucleotides (containing D-ribose) , including tRNA, rRNA, hRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N-or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acid ( "PNA” ) ) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, OR.
  • PNA peptide nucleic acid
  • these terms include, for example, 3'-deoxy-2', 5'-DNA, oligodeoxyribonucleotide N3'to P5'phosphoramidates, 2'-O-alkyl-substituted RNA, hybrids between DNA and RNA or between PNAs and DNA or RNA, and also include known types of modifications, for example, labels, alkylation, "caps, " substitution of one or more of the nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • modifications for example, labels, alkylation, "caps, " substitution of one or more of the nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc
  • linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • positively charged linkages e.g., aminoalkylphosphoramidates, aminoalkylphosphotriesters
  • pendant moieties such as, for example, proteins (including enzymes (e.g. nucleases) , toxins, antibodies, signal peptides, poly-L-lysine, etc. ) , those with intercalators (e.g., acridine, psoralen, etc. ) , those containing chelates (of, e.g., metals, radioactive metals, boron, oxidative metals, etc.
  • nucleoside and nucleotide will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides can also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or are functionalized as ethers, amines, or the like.
  • the term “nucleotidic unit” is intended to encompass nucleosides and nucleotides.
  • Nucleic acid probe and “probe” are used interchangeably and refer to a structure comprising a polynucleotide, as defined above, that contains a nucleic acid sequence that can bind to a corresponding target.
  • the polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.
  • complementary or matched means that two nucleic acid sequences have at least 50%sequence identity. Preferably, the two nucleic acid sequences have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%or 100%of sequence identity. “Complementary or matched” also means that two nucleic acid sequences can hybridize under low, middle and/or high stringency condition (s) . The percentage of sequence identity or homology is calculated by comparing one to another when aligned to corresponding portions of the reference sequence.
  • substantially complementary or substantially matched means that two nucleic acid sequences have at least 90%sequence identity. Preferably, the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99%or 100%of sequence identity. Alternatively, “substantially complementary or substantially matched” means that two nucleic acid sequences can hybridize under high stringency condition (s) . The percentage of sequence identity or homology is calculated by comparing one to another when aligned to corresponding portions of the reference sequence.
  • complementary and substantially complementary include the hybridization or base pairing or the formation of a duplex between nucleotides or nucleic acids, for instance, between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid.
  • Complementary nucleotides are, generally, A and T (or A and U) , or C and G.
  • Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80%of the other strand, usually at least about 90%to about 95%, and even about 98%to about 100%.
  • two complementary sequences of nucleotides are capable of hybridizing, preferably with less than 25%, more preferably with less than 15%, even more preferably with less than 5%, most preferably with no mismatches between opposed nucleotides.
  • the two molecules will hybridize under conditions of high stringency.
  • Hybridization as used herein may refer to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide.
  • the resulting double-stranded polynucleotide can be a “hybrid” or “duplex.
  • “Hybridization conditions” typically include salt concentrations of approximately less than 1 M, often less than about 500 mM and may be less than about 200 mM.
  • a “hybridization buffer” includes a buffered salt solution such as 5%SSPE, or other such buffers known in the art.
  • Hybridization temperatures can be as low as 5°C, but are typically greater than 22°C, and more typically greater than about 30°C, and typically in excess of 37°C.
  • Hybridizations are often performed under stringent conditions, i.e., conditions under which a sequence will hybridize to its target sequence but will not hybridize to other, non-complementary sequences. Stringent conditions are sequence-dependent and are different in different circumstances. For example, longer fragments may require higher hybridization temperatures for specific hybridization than short fragments. As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents, and the extent of base mismatching, the combination of parameters is more important than the absolute measure of any one parameter alone.
  • Tm can be the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • Tm can be the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • the stability of a hybrid is a function of the ion concentration and temperature.
  • a hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency.
  • Exemplary stringent conditions include a salt concentration of at least 0.01 M to no more than 1 M sodium ion concentration (or other salt) at a pH of about 7.0 to about 8.3 and a temperature of at least 25°C.
  • conditions of 5 ⁇ SSPE 750 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA at pH 7.4
  • a temperature of approximately 30°C are suitable for allele-specific hybridizations, though a suitable temperature depends on the length and/or GC content of the region hybridized.
  • “stringency of hybridization” in determining percentage mismatch can be as follows: 1) high stringency: 0.1 ⁇ SSPE, 0.1%SDS, 65°C; 2) medium stringency: 0.2 ⁇ SSPE, 0.1%SDS, 50°C (also referred to as moderate stringency) ; and 3) low stringency: 1.0 ⁇ SSPE, 0.1%SDS, 50°C. It is understood that equivalent stringencies may be achieved using alternative buffers, salts and temperatures.
  • moderately stringent hybridization can refer to conditions that permit a nucleic acid molecule such as a probe to bind a complementary nucleic acid molecule.
  • the hybridized nucleic acid molecules generally have at least 60%identity, including for example at least any of 70%, 75%, 80%, 85%, 90%, or 95%identity.
  • Moderately stringent conditions can be conditions equivalent to hybridization in 50%formamide, 5 ⁇ Denhardt’s solution, 5x SSPE, 0.2%SDS at 42°C, followed by washing in 0.2 ⁇ SSPE, 0.2%SDS, at 42°C.
  • High stringency conditions can be provided, for example, by hybridization in 50%formamide, 5 ⁇ Denhardt’s solution, 5 ⁇ SSPE, 0.2%SDS at 42°C, followed by washing in 0.1 ⁇ SSPE, and 0.1%SDS at 65°C.
  • Low stringency hybridization can refer to conditions equivalent to hybridization in 10%formamide, 5 ⁇ Denhardt’s solution, 6 ⁇ SSPE, 0.2%SDS at 22°C, followed by washing in 1x SSPE, 0.2%SDS, at 37°C.
  • Denhardt’s solution contains 1%Ficoll, 1%polyvinylpyrolidone, and 1%bovine serum albumin (BSA) .
  • BSA bovine serum albumin
  • 20 ⁇ SSPE sodium chloride, sodium phosphate, EDTA
  • RNA or DNA strand will hybridize under selective hybridization conditions to its complement.
  • selective hybridization will occur when there is at least about 65%complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90%complementary. See M. Kanehisa, Nucleic Acids Res. 12: 203 (1984) .
  • homologous denotes a sequence of amino acids having at least 50%, 60%, 70%, 80%or 90%identity wherein one sequence is compared to a reference sequence of amino acids. The percentage of sequence identity or homology is calculated by comparing one to another when aligned to corresponding portions of the reference sequence.
  • a “primer” used herein can be an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3'end along the template so that an extended duplex is formed.
  • the sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide.
  • Primers usually are extended by a polymerase, for example, a DNA polymerase.
  • Loop-mediated amplification is a DNA amplification technique developed by Notomi et al. Notomi etal., 2000, “Loop-mediated isothermal amplification of DNA, ” Nucleic Acids Res. 28 (12) : E63.
  • LAMP the target nucleic acid sequence is typically amplified at a constant temperature between about 60°C and about 65°C using either two or three pairs of primers and a polymerase with high-strand displacement activity in addition to a replication activity.
  • the loop-mediated isothermal amplification (LAMP) reaction is a highly specific, sensitive, isothermal nucleic acid amplification reaction.
  • LAMP employs a primer set of four essential primers, termed forward inner primer (FIP) , backward inner primer (BIP) , forward displacement primer (F3) , and backward displacement primer (B3) .
  • FIP forward inner primer
  • BIP backward inner primer
  • F3 forward displacement primer
  • B3 backward displacement primer
  • B3 backward displacement primer
  • LF forward loop primer
  • B3 backward loop primer
  • LF forward loop primer
  • LB backward loop primer
  • the inner primers contain sequences of the sense and antisense strands of the target DNA, while each of the displacement primers (F3 and B3) and the loop primers (LF and LB) contains a single target sequence. In total, eight target sequences are recognized when including loop primers (LF and LB) in the reaction.
  • a DNA polymerase is used to amplify the target sequence of interest. Many different DNA polymerases may be used, the most common being the Bst DNA polymerase while the Geobacillus sp. large fragment (GspSSD) DNA polymerase is used less often.
  • the LAMP reaction is initiated by DNA synthesis primed by the inner primers (FIP and BIP) .
  • DNA synthesis primed by a displacement primer F3 or B3 which releases a single-stranded DNA.
  • This single-stranded DNA serves as template for DNA synthesis primed by the second inner and displacement primers that hybridize to the other end of the target.
  • one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis. This yields the original stem-loop DNA and a new stem-loop DNA with a stem twice as long.
  • the cycling reaction continues with accumulation of typically around 10 9 copies of target in less than an hour.
  • Non-specific target detection may be obtained through visual identification of a turbid sample as magnesium pyrophosphate precipitates in a positive LAMP reaction.
  • various agents such as hydroxy naphthol blue or calcein, may be added to the reaction.
  • fluorescent detection may be achieved using a DNA intercalating dye, such as SYBR green, Picogreen or propedium iodide, which is added to the reaction reagent or added after the completion of the reaction for end point analysis.
  • Detecting the amplification of a specific target of interest by the LAMP reaction may be achieved using various hybridization probe-based methods. For example, by labelling a loop primer with a fluorophore and adding a DNA probe which is complementary to the loop primer but labelled with a quencher molecule. Initially, the loop primer will bind the DNA probe, and the fluorophore will not emit light. When the LAMP reaction proceeds, the loop primer is incorporated into the stem-loop structure and the DNA probe cannot quench the fluorophore resulting in a fluorescent signal. LAMP products may also readily be identified using gel electrophoresis which visualizes distinct banding patterns depending on the specific target and primers used. The technique is well-known to the skilled person and described in detail, e.g., in Nagamine et al., Mol. Cell. Probes (2002) 16, 223-229.
  • loop-mediated isothermal amplification uses a Bst DNA polymerase having a strand displacement activity and a waterfall nucleic acid amplification feature.
  • LAMP is performed under isothermal conditions to denature and amplify nucleic acids in automatic cycles.
  • the strand displacement nucleic acid amplification reaction, nucleic acid denaturation, and automatic cycle chain replacement occur under isothermal conditions.
  • LAMP typically uses four specific primers for six regions of the target sequence, and a strand displacement DNA polymerase or polymerases are used at a constant temperature to constantly replicate and amplify DNA.
  • the reaction system may further comprise two loop primers, which are allowed to bind the stem-loop structure and to start the strand displacement synthesis and repeat the reaction cycle.
  • LAMP is a simple, rapid, sensitive, and specific reaction, and is especially suitable for designing and manufacturing detection kits based on the technology.
  • the LAMP primers are the key factors in the sensitivity and specificity of the test results using the LAMP technology.
  • the technical problem to be solved is how to detect respiratory tract infection pathogens species.
  • a system for the detection of pathogenic organisms related to respiratory infections for example, a system comprising dedicated LAMP primers specific for amplification and detection of a nucleic acid sequence from the pathogens.
  • LAMP loop-mediated isothermal amplification
  • the primer sets include all thirteen, any twelve sets, any eleven sets, any ten sets, any nine sets, any eight sets, any seven sets, any six sets, any five sets, any four sets, any three sets, any two sets, or any one set selected from the group consisting of: the primer set for detecting Streptococcus pneumoniae (SP) , the primer set for detecting Staphylococcus aureus (SA) , the primer set for detecting methicillin-resistant Staphylococcus aureus (MRSA) , the primer set for detecting Escherichia coli (E.
  • SP Streptococcus pneumoniae
  • SA Staphylococcus aureus
  • MRSA methicillin-resistant Staphylococcus aureus
  • E Escherichia coli
  • the primer set for detecting Klebsiella pneumoniae (KP) the primer set for detecting Pseudomonas aeruginosa (PA) , the primer set for detecting Acinetobacter baumannii (AB) , the primer set for detecting Stenotrophomonas maltophilia (SMA) , the primer set for detecting Haemophilus influenzae (HI) , the primer set for detecting Legionella pneumophila (LP) , the primer set for detecting Mycobacterium tuberculosis complex (MTC) , the primer set for detecting Mycoplasma pneumoniae (MP) , and the primer set for detecting Chlamydia pneumoniae (CP) .
  • KP Klebsiella pneumoniae
  • PA Pseudomonas aeruginosa
  • AB Acinetobacter baumannii
  • SMA Stenotrophomonas maltophilia
  • HI Haemophilus influenzae
  • LP the
  • the Streptococcus pneumoniae (SP) primer set can be a primer set comprising, consisting essentially of, or consisting of six sense primers for SP (e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP) , a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for SP (e.g., primers comprising the reverse-complementary sequences of the six sense primers) , a primer set comprising, consisting essentially of, or consisting of four sense primers for SP (e.g., the F3, B3, FIP, and BIP primers for LAMP) , or a primer set comprising, consisting essentially of, or consisting of four anti-sense primers for SP (e.g., primers comprising the reverse-complementary sequences of the four sense primers) .
  • six sense primers for SP e.g., the F3, B3, FIP, BIP, LF, and
  • the Streptococcus pneumoniae (SP) primer set comprises SP primer F3-Z, SP primer B3-Z, SP primer FIP-Z, and SP primer BIP-Z, and optionally SP primer LF-Z and SP primer LB-Z, for example, as shown in Table 1.
  • the SP primer set comprises SP primer F3-F, SP primer B3-F, SP primer FIP-F, and SP primer BIP-F, and optionally SP primer LF-F and SP primer LB-F, for example, as shown in Table 2.
  • the SP primer F3-Z in the sets of primers provided herein is either of the single-stranded DNA in a1) or a2) as follows:
  • a1 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 6-25 of SEQ ID NO: 1;
  • a2) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of a1) .
  • the SP primer B3-Z in the sets of primers provided herein is either of the single-stranded DNA in a4) or a5) as follows:
  • a4 a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 213-229 of SEQ ID NO: 1;
  • the SP primer FIP-Z in the sets of primers provided herein is either of the single-stranded DNA in a7) or a8) as follows:
  • a7 a single-stranded DNA of the formula A-linker-B, A comprising, consisting essentially of, or consisting of the reverse-complementary sequence of the single-stranded DNA at positions 79-100 of SEQ ID NO: 1; the linker comprising, consisting essentially of, or consisting of a single-stranded DNA of between about 0 and about 10 nucleotides in length; and B comprising, consisting essentially of, or consisting of the sequence of the single-stranded DNA at positions 39-55 of SEQ ID NO: 1;
  • a8 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of a7) .
  • the SP primer BIP-Z in the sets of primers provided herein is either of the single-stranded DNA in a10) or a11) as follows:
  • A-linker-B A comprising, consisting essentially of, or consisting of the sequence of the single-stranded DNA at positions 101-124 of SEQ ID NO: 1; the linker comprising, consisting essentially of, or consisting of a single-stranded DNA of between about 0 and about 10 nucleotides in length; and B comprising, consisting essentially of, or consisting of the reverse-complementary sequence of the single-stranded DNA at positions 166-185 of SEQ ID NO: 1;
  • a11 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of a10) .
  • the SP primer LF-Z in the sets of primers provided herein is either of the single-stranded DNA in a13) or a14) as follows:
  • a13) a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 56-77 of SEQ ID NO: 1;
  • a14 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of a13) .
  • the SP primer LB-Z in the sets of primers provided herein is either of the single-stranded DNA in a16) or a17) as follows:
  • a16 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 126-146 of SEQ ID NO: 1;
  • a17 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of a16) .
  • the SP primer F3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SP primer F3-Z.
  • the SP primer B3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SP primer B3-Z.
  • the SP primer FIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SP primer FIP-Z.
  • the SP primer BIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SP primer BIP-Z.
  • the SP primer LF-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SP primer LF-Z. In one aspect, the SP primer LB-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SP primer LB-Z.
  • the Staphylococcus aureus (SA) primer set can be a primer set comprising, consisting essentially of, or consisting of six sense primers for SA (e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP) , a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for SA (e.g., primers comprising the reverse-complementary sequences of the six sense primers) , a primer set comprising, consisting essentially of, or consisting of four sense primers for SA (e.g., the F3, B3, FIP, and BIP primers for LAMP) , or a primer set comprising, consisting essentially of, or consisting of four anti-sense primers for SA (e.g., primers comprising the reverse-complementary sequences of the four sense primers) .
  • six sense primers for SA e.g., the F3, B3, FIP, BIP, LF,
  • the Staphylococcus aureus (SA) primer set comprises SA primer F3-Z, SA primer B3-Z, SA primer FIP-Z, and SA primer BIP-Z, and optionally SA primer LF-Z and SA primer LB-Z, for example, as shown in Table 1.
  • the SA primer set comprises SA primer F3-F, SA primer B3-F, SA primer FIP-F, and SA primer BIP-F, and optionally SA primer LF-F and SA primer LB-F, for example, as shown in Table 2.
  • the SA primer F3-Z in the sets of primers provided herein is either of the single-stranded DNA in b1) or b2) as follows:
  • b1) a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 11-30 of SEQ ID NO: 2;
  • b2) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of b1) .
  • the SA primer B3-Z in the sets of primers provided herein is either of the single-stranded DNA in b4) or b5) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 222-243 of SEQ ID NO: 2;
  • b5) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of b4) .
  • the SA primer FIP-Z in the sets of primers provided herein is either of the single-stranded DNA in b7) or b8) as follows:
  • A-linker-B A comprising, consisting essentially of, or consisting of the reverse-complementary sequence of the single-stranded DNA at positions 72-95 of SEQ ID NO: 2; the linker comprising, consisting essentially of, or consisting of a single-stranded DNA of between about 0 and about 10 nucleotides in length; and B comprising, consisting essentially of, or consisting of the sequence of the single-stranded DNA at positions 31-50 of SEQ ID NO: 2;
  • b8) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of b7) .
  • the SA primer BIP-Z in the sets of primers provided herein is either of the single-stranded DNA in b10) or b11) as follows:
  • A-linker-B A comprising, consisting essentially of, or consisting of the sequence of the single-stranded DNA at positions 127-149 of SEQ ID NO: 2; the linker comprising, consisting essentially of, or consisting of a single-stranded DNA of between about 0 and about 10 nucleotides in length; and B comprising, consisting essentially of, or consisting of the reverse-complementary sequence of the single-stranded DNA at positions 196-212 of SEQ ID NO: 2;
  • b11) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of b10) .
  • the SA primer LF-Z in the sets of primers provided herein is either of the single-stranded DNA in b13) or b14) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 54-71 of SEQ ID NO: 2;
  • b14 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of b13) .
  • the SA primer LB-Z in the sets of primers provided herein is either of the single-stranded DNA in b16) or b17) as follows:
  • b16 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 157-178 of SEQ ID NO: 2;
  • b17 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of b16) .
  • the SA primer F3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SA primer F3-Z.
  • the SA primer B3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SA primer B3-Z.
  • the SA primer FIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SA primer FIP-Z.
  • the SA primer BIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SA primer BIP-Z.
  • the SA primer LF-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SA primer LF-Z. In one aspect, the SA primer LB-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SA primer LB-Z.
  • the methicillin-resistant Staphylococcus aureus (MRSA) primer set can be a primer set comprising, consisting essentially of, or consisting of six sense primers for MRSA (e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP) , a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for MRSA (e.g., primers comprising the reverse-complementary sequences of the six sense primers) , a primer set comprising, consisting essentially of, or consisting of four sense primers for MRSA (e.g., the F3, B3, FIP, and BIP primers for LAMP) , or a primer set comprising, consisting essentially of, or consisting of four anti-sense primers for MRSA (e.g., primers comprising the reverse-complementary sequences of the four sense primers) .
  • MRSA methicillin-resistant Staphylococcus aureus
  • the methicillin-resistant Staphylococcus aureus (MRSA) primer set comprises MRSA primer F3-Z, MRSA primer B3-Z, MRSA primer FIP-Z, and MRSA primer BIP-Z, and optionally MRSA primer LF-Z and MRSA primer LB-Z, for example, as shown in Table 1.
  • the MRSA primer set comprises MRSA primer F3-F, MRSA primer B3-F, MRSA primer FIP-F, and MRSA primer BIP-F, and optionally MRSA primer LF-F and MRSA primer LB-F, for example, as shown in Table 2.
  • the MRSA primer F3-Z in the sets of primers provided herein is either of the single-stranded DNA in c1) or c2) as follows:
  • c1) a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 12-30 of SEQ ID NO: 3;
  • c2) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of c1) .
  • the MRSA primer B3-Z in the sets of primers provided herein is either of the single-stranded DNA in c4) or c5) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 211-234 of SEQ ID NO: 3;
  • c5) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of c4) .
  • the MRSA primer FIP-Z in the sets of primers provided herein is either of the single-stranded DNA in c7) or c8) as follows:
  • A-linker-B in which A comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 97-121 of SEQ ID NO: 3, the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length, and B comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 47-71 of SEQ ID NO: 3;
  • c8) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of c7) .
  • the MRSA primer BIP-Z in the sets of primers provided herein is either of the single-stranded DNA in c10) or c11) as follows:
  • A-linker-B in which A comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 133-156 of SEQ ID NO: 3, the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length, and B comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 182-206 of SEQ ID NO: 3;
  • c11 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of c10) .
  • the MRSA primer LF-Z in the sets of primers provided herein is either of the single-stranded DNA in c13) or c14) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 72-95 of SEQ ID NO: 3;
  • c14 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of c13) .
  • the MRSA primer LB-Z in the sets of primers provided herein is either of the single-stranded DNA in c16) or c17) as follows:
  • c16 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 157-181 of SEQ ID NO: 3;
  • c17 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of c16) .
  • the MRSA primer F3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MRSA primer F3-Z. In one aspect, the MRSA primer B3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MRSA primer B3-Z. In one aspect, the MRSA primer FIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MRSA primer FIP-Z. In one aspect, the MRSA primer BIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MRSA primer BIP-Z.
  • the MRSA primer LF-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MRSA primer LF-Z. In one aspect, the MRSA primer LB-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MRSA primer LB-Z.
  • the E. coli primer set can be a primer set comprising, consisting essentially of, or consisting of six sense primers for E. coli (e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP) , a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for E. coli (e.g., primers comprising the reverse-complementary sequences of the six sense primers) , a primer set comprising, consisting essentially of, or consisting of four sense primers for E.
  • six sense primers for E. coli e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP
  • a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for E. coli (e.g., primers comprising the reverse-complementary sequences of the six sense primers)
  • a primer set comprising, consisting essentially
  • coli e.g., the F3, B3, FIP, and BIP primers for LAMP
  • a primer set comprising, consisting essentially of, or consisting of four anti-sense primers for E. coli (e.g., primers comprising the reverse-complementary sequences of the four sense primers) .
  • the E. coli primer set comprises E. coli primer F3-Z, E. coli primer B3-Z, E. coli primer FIP-Z, and E. coli primer BIP-Z, and optionally E. coli primer LF-Z and E. coli primer LB-Z, for example, as shown in Table 1.
  • the E. coli primer set comprises E. coli primer F3-F, E. coli primer B3-F, E. coli primer FIP-F, and E. coli primer BIP-F, and optionally E. coli primer LF-F and E. coli primer LB-F, for example, as shown in Table 2.
  • the E. coli primer F3-Z in the sets of primers provided herein is either of the single-stranded DNA in d1) or d2) as follows:
  • d1) a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 11-30 of SEQ ID NO: 4;
  • d2) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of d1) .
  • the E. coli primer B3-Z in the sets of primers provided herein is either of the single-stranded DNA in d4) or d5) as follows:
  • d4) a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 200-219 of SEQ ID NO: 4;
  • d5) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of d4) .
  • the E. coli primer FIP-Z in the sets of primers provided herein is either of the single-stranded DNA in d7) or d8) as follows:
  • A-linker-B in which A comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 77-96 of SEQ ID NO: 4, the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length, and B comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 38-55 of SEQ ID NO: 4;
  • d8) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of d7) .
  • the E. coli primer BIP-Z in the sets of primers provided herein is either of the single-stranded DNA in d10) or d11) as follows:
  • A-linker-B a single-stranded DNA of the formula A-linker-B, in which A comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 101-122 of SEQ ID NO: 4, the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length, and B comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 163-183 of SEQ ID NO: 4;
  • d11 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of d10) .
  • the E. coli primer LF-Z in the sets of primers provided herein is either of the single-stranded DNA in d13) or d14) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 56-76 of SEQ ID NO: 4;
  • d14 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of d13) .
  • the E. coli primer LB-Z in the sets of primers provided herein is either of the single-stranded DNA in d16) or d17) as follows:
  • d16 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 137-154 of SEQ ID NO: 4;
  • d17 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of d16) .
  • the E. coli primer F3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the E. coli primer F3-Z.
  • the E. coli primer B3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the E. coli primer B3-Z.
  • the E. coli primer FIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the E. coli primer FIP-Z.
  • the E. coli primer BIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the E. coli primer BIP-Z.
  • the E. coli primer LF-F comprises, consists essentially of, or consists of the reverse complementary sequence of the E. coli primer LF-Z.
  • the E. coli primer LB-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the E. coli primer LB-Z.
  • the Klebsiella pneumoniae (KP) primer set can be a primer set comprising, consisting essentially of, or consisting of six sense primers for KP (e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP) , a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for KP (e.g., primers comprising the reverse-complementary sequences of the six sense primers) , a primer set comprising, consisting essentially of, or consisting of four sense primers for KP (e.g., the F3, B3, FIP, and BIP primers for LAMP) , or a primer set comprising, consisting essentially of, or consisting of four anti-sense primers for KP (e.g., primers comprising the reverse-complementary sequences of the four sense primers) .
  • six sense primers for KP e.g., the F3, B3, FIP, BIP
  • the KP primer set comprises KP primer F3-Z, KP primer B3-Z, KP primer FIP-Z, and KP primer BIP-Z, and optionally KP primer LF-Z and KP primer LB-Z, for example, as shown in Table 1.
  • the KP primer set comprises KP primer F3-F, KP primer B3-F, KP primer FIP-F, and KP primer BIP-F, and optionally KP primer LF-F and KP primer LB-F, for example, as shown in Table 2.
  • the KP primer F3-Z in the sets of primers provided herein is either of the single-stranded DNA in e1) or e2) as follows:
  • e1 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 14-30 of SEQ ID NO: 5;
  • e2) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of e1) .
  • the KP primer B3-Z in the sets of primers provided herein is either of the single-stranded DNA in e4) or e5) as follows:
  • e4 a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 186-203 of SEQ ID NO: 5;
  • e5 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of e4) .
  • the KP primer FIP-Z in the sets of primers provided herein is either of the single-stranded DNA in e7) or e8) as follows:
  • A-linker-B in which A comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 90-108 of SEQ ID NO: 5, the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length, and B comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 38-54 of SEQ ID NO: 5;
  • e8) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of e7) .
  • the KP primer BIP-Z in the sets of primers provided herein is either of the single-stranded DNA in e10) or e11) as follows:
  • A-linker-B in which A comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 118-140 of SEQ ID NO: 5, the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length, and B comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 168-185 of SEQ ID NO: 5;
  • e11 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of e10) .
  • the KP primer LF-Z in the sets of primers provided herein is either of the single-stranded DNA in e13) or e14) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 68-88 of SEQ ID NO: 5;
  • e14 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of e13) .
  • the KP primer LB-Z in the sets of primers provided herein is either of the single-stranded DNA in e16) or e17) as follows:
  • e16 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 151-167 of SEQ ID NO: 5;
  • e17 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of e16) .
  • the KP primer F3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the KP primer F3-Z. In one aspect, the KP primer B3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the KP primer B3-Z. In one aspect, the KP primer FIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the KP primer FIP-Z. In one aspect, the KP primer BIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the KP primer BIP-Z.
  • the KP primer LF-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the KP primer LF-Z.
  • the KP primer LB-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the KP primer LB-Z.
  • the Pseudomonas aeruginosa (PA) primer set can be a primer set comprising, consisting essentially of, or consisting of six sense primers for PA (e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP) , a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for PA (e.g., primers comprising the reverse-complementary sequences of the six sense primers) , a primer set comprising, consisting essentially of, or consisting of four sense primers for PA (e.g., the F3, B3, FIP, and BIP primers for LAMP) , or a primer set comprising, consisting essentially of, or consisting of four anti-sense primers for PA (e.g., primers comprising the reverse-complementary sequences of the four sense primers) .
  • the PA primer set comprises PA primer F3-Z, PA primer B3-Z, PA primer FIP-Z, and PA primer BIP-Z, and optionally PA primer LF-Z and PA primer LB-Z, for example, as shown in Table 1.
  • the PA primer set comprises PA primer F3-F, PA primer B3-F, PA primer FIP-F, and PA primer BIP-F, and optionally PA primer LF-F and PA primer LB-F, for example, as shown in Table 2.
  • the PA primer F3-Z in the sets of primers provided herein is either of the single-stranded DNA in f1) or f2) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 11-30 of SEQ ID NO: 6;
  • f2) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of f1) .
  • the PA primer B3-Z in the sets of primers provided herein is either of the single-stranded DNA in f4) or f5) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 218-235 of SEQ ID NO: 6;
  • f5) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of f4) .
  • the PA primer FIP-Z in the sets of primers provided herein is either of the single-stranded DNA in f7) or f8) as follows:
  • A-linker-B in which A comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 75-93 of SEQ ID NO: 6, the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length, and B comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 35-53 of SEQ ID NO: 6;
  • f8) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of f7) .
  • the PA primer BIP-Z in the sets of primers provided herein is either of the single-stranded DNA in f10) or f11) as follows:
  • A-linker-B comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 126-144 of SEQ ID NO: 6,
  • the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length
  • B comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 188-205 of SEQ ID NO: 6;
  • f11 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of f10) .
  • the PA primer LF-Z in the sets of primers provided herein is either of the single-stranded DNA in f13) or f14) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 54-73 of SEQ ID NO: 6;
  • f14 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of f13) .
  • the PA primer LB-Z in the sets of primers provided herein is either of the single-stranded DNA in f16) or f17) as follows:
  • f17 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of f16) .
  • the PA primer F3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the PA primer F3-Z.
  • the PA primer B3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the PA primer B3-Z.
  • the PA primer FIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the PA primer FIP-Z.
  • the PA primer BIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the PA primer BIP-Z.
  • the PA primer LF-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the PA primer LF-Z. In one aspect, the PA primer LB-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the PA primer LB-Z.
  • the Acinetobacter baumannii (AB) primer set can be a primer set comprising, consisting essentially of, or consisting of six sense primers for AB (e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP) , a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for AB (e.g., primers comprising the reverse-complementary sequences of the six sense primers) , a primer set comprising, consisting essentially of, or consisting of four sense primers for AB (e.g., the F3, B3, FIP, and BIP primers for LAMP) , or a primer set comprising, consisting essentially of, or consisting of four anti-sense primers for AB (e.g., primers comprising the reverse-complementary sequences of the four sense primers) .
  • six sense primers for AB e.g., the F3, B3, FIP, BIP
  • the AB primer set comprises AB primer F3-Z, AB primer B3-Z, AB primer FIP-Z, and AB primer BIP-Z, and optionally AB primer LF-Z and AB primer LB-Z, for example, as shown in Table 1.
  • the AB primer set comprises AB primer F3-F, AB primer B3-F, AB primer FIP-F, and AB primer BIP-F, and optionally AB primer LF-F and AB primer LB-F, for example, as shown in Table 2.
  • the AB primer F3-Z in the sets of primers provided herein is either of the single-stranded DNA in g1) or g2) as follows:
  • g1 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 10-30 of SEQ ID NO: 7;
  • g2) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of g1) .
  • the AB primer B3-Z in the sets of primers provided herein is either of the single-stranded DNA in g4) or g5) as follows:
  • g4) a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 206-227 of SEQ ID NO: 7;
  • g5 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of g4) .
  • the AB primer FIP-Z in the sets of primers provided herein is either of the single-stranded DNA in g7) or g8) as follows:
  • A-linker-B in which A comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 73-94 of SEQ ID NO: 7, the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length, and B comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 31-55 of SEQ ID NO: 7;
  • g8 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of g7) .
  • the AB primer BIP-Z in the sets of primers provided herein is either of the single-stranded DNA in g10) or g11) as follows:
  • A-linker-B in which A comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 128-152 of SEQ ID NO: 7, the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length, and B comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 186-204 of SEQ ID NO: 7;
  • g11 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of g10) .
  • the AB primer LF-Z in the sets of primers provided herein is either of the single-stranded DNA in g13) or g14) as follows:
  • g13 a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 56-71 ofSEQ ID NO: 7;
  • g14 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of g13) .
  • the AB primer LB-Z in the sets of primers provided herein is either of the single-stranded DNA in g16) or g17) as follows:
  • g16 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 156-173 of SEQ ID NO: 7;
  • g17 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of g16) .
  • the AB primer F3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the AB primer F3-Z. In one aspect, the AB primer B3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the AB primer B3-Z. In one aspect, the AB primer FIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the AB primer FIP-Z. In one aspect, the AB primer BIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the AB primer BIP-Z.
  • the AB primer LF-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the AB primer LF-Z. In one aspect, the AB primer LB-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the AB primer LB-Z.
  • the Stenotrophomonas maltophilia (SMA) primer set can be a primer set comprising, consisting essentially of, or consisting of six sense primers for SMA (e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP) , a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for SMA (e.g., primers comprising the reverse-complementary sequences of the six sense primers) , a primer set comprising, consisting essentially of, or consisting of four sense primers for SMA (e.g., the F3, B3, FIP, and BIP primers for LAMP) , or a primer set comprising, consisting essentially of, or consisting of four anti-sense primers for SMA (e.g., primers comprising the reverse-complementary sequences of the four sense primers) .
  • six sense primers for SMA e.g., the F3, B
  • the SMA primer set comprises SMA primer F3-Z, SMA primer B3-Z, SMA primer FIP-Z, and SMA primer BIP-Z, and optionally SMA primer LF-Z and SMA primer LB-Z, for example, as shown in Table 1.
  • the SMA primer set comprises SMA primer F3-F, SMA primer B3-F, SMA primer FIP-F, and SMA primer BIP-F, and optionally SMA primer LF-F and SMA primer LB-F, for example, as shown in Table 2.
  • the SMA primer F3-Z in the sets of primers provided herein is either of the single-stranded DNA in h1) or h2) as follows:
  • h1 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 11-30 of SEQ ID NO: 8;
  • h2) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of h1) .
  • the SMA primer B3-Z in the sets of primers provided herein is either of the single-stranded DNA in h4) or h5) as follows:
  • h4 a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 197-213 of SEQ ID NO: 8;
  • h5 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of h4) .
  • the SMA primer FIP-Z in the sets of primers provided herein is either of the single-stranded DNA in h7) or h8) as follows:
  • A-linker-B in which A comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 81-100 of SEQ ID NO: 8, the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length, and B comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 41-57 of SEQ ID NO: 8;
  • h8 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of h7) .
  • the SMA primer BIP-Z in the sets of primers provided herein is either of the single-stranded DNA in h10) or h11) as follows:
  • A-linker-B in which A comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 109-132 of SEQ ID NO: 8, the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length, and B comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 175-193 of SEQ ID NO: 8;
  • h11 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of h10) .
  • the SMA primer LF-Z in the sets of primers provided herein is either of the single-stranded DNA in h13) or h14) as follows:
  • h13 a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 58-73 of SEQ ID NO: 8;
  • h14 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of h13) .
  • the SMA primer LB-Z in the sets of primers provided herein is either of the single-stranded DNA in h16) or h17) as follows:
  • h16 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 136-153 of SEQ ID NO: 8;
  • h17 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of h16) .
  • the SMA primer F3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SMA primer F3-Z.
  • the SMA primer B3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SMA primer B3-Z.
  • the SMA primer FIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SMA primer FIP-Z.
  • the SMA primer BIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SMA primer BIP-Z.
  • the SMA primer LF-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SMA primer LF-Z. In one aspect, the SMA primer LB-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the SMA primer LB-Z.
  • the Haemophilus influenzae (HI) primer set can be a primer set comprising, consisting essentially of, or consisting of six sense primers for HI (e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP) , a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for HI (e.g., primers comprising the reverse-complementary sequences of the six sense primers) , a primer set comprising, consisting essentially of, or consisting of four sense primers for HI (e.g., the F3, B3, FIP, and BIP primers for LAMP) , or a primer set comprising, consisting essentially of, or consisting of four anti-sense primers for HI (e.g., primers comprising the reverse-complementary sequences of the four sense primers) .
  • six sense primers for HI e.g., the F3, B3, FIP, BIP,
  • the HI primer set comprises HI primer F3-Z, HI primer B3-Z, HI primer FIP-Z, and HI primer BIP-Z, and optionally HI primer LF-Z and HI primer LB-Z, for example, as shown in Table 1.
  • the HI primer set comprises HI primer F3-F, HI primer B3-F, HI primer FIP-F, and HI primer BIP-F, and optionally HI primer LF-F and HI primer LB-F, for example, as shown in Table 2.
  • the HI primer F3-Z in the sets of primers provided herein is either of the single-stranded DNA in i1) or i2) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 12-30 of SEQ ID NO: 9;
  • i2) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of i1) .
  • the HI primer B3-Z in the sets of primers provided herein is either of the single-stranded DNA in i4) or i5) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 223-246 of SEQ ID NO: 9;
  • a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of i4) .
  • the HI primer FIP-Z in the sets of primers provided herein is either of the single-stranded DNA in i7) or i8) as follows:
  • A-linker-B in which A comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 83-107 of SEQ ID NO: 9, the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length, and B comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 39-58 of SEQ ID NO: 9;
  • the HI primer BIP-Z in the sets of primers provided herein is either of the single-stranded DNA in i10) or i11) as follows:
  • A-linker-B in which A comprises, consists essentially of, or consists of the sequence of the single-stranded DNA at positions 114-139 of SEQ ID NO: 9, the linker comprises, consists essentially of, or consists of a single-stranded DNA of between about 0 and about 10 nucleotides in length, and B comprises, consists essentially of, or consists of the reverse-complementary sequence of the single-stranded DNA at positions 176-199 of SEQ ID NO: 9;
  • i11 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of i10) .
  • the HI primer LF-Z in the sets of primers provided herein is either of the single-stranded DNA in i13) or i14) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 62-82 of SEQ ID NO: 9;
  • the HI primer LB-Z in the sets of primers provided herein is either of the single-stranded DNA in i16) or i17) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 141-161 of SEQ ID NO: 9;
  • i17 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of i16) .
  • the HI primer F3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the HI primer F3-Z.
  • the HI primer B3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the HI primer B3-Z.
  • the HI primer FIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the HI primer FIP-Z.
  • the HI primer BIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the HI primer BIP-Z.
  • the HI primer LF-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the HI primer LF-Z.
  • the HI primer LB-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the HI primer LB-Z.
  • the Legionella pneumophila (LP) primer set can be a primer set comprising, consisting essentially of, or consisting of six sense primers for LP (e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP) , a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for LP (e.g., primers comprising the reverse-complementary sequences of the six sense primers) , a primer set comprising, consisting essentially of, or consisting of four sense primers for LP (e.g., the F3, B3, FIP, and BIP primers for LAMP) , or a primer set comprising, consisting essentially of, or consisting of four anti-sense primers for LP (e.g., primers comprising the reverse-complementary sequences of the four sense primers) .
  • six sense primers for LP e.g., the F3, B3, FIP, BIP,
  • the LP primer set comprises LP primer F3-Z, LP primer B3-Z, LP primer FIP-Z, and LP primer BIP-Z, and optionally LP primer LF-Z and LP primer LB-Z, for example, as shown in Table 1.
  • the LP primer set comprises LP primer F3-F, LP primer B3-F, LP primer FIP-F, and LP primer BIP-F, and optionally LP primer LF-F and LP primer LB-F, for example, as shown in Table 2.
  • the LP primer F3-Z in the sets of primers provided herein is either of the single-stranded DNA in j1) or j2) as follows:
  • j1) a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 10-30 of SEQ ID NO: 10;
  • j2) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of j1) .
  • the LP primer B3-Z in the sets of primers provided herein is either of the single-stranded DNA in j4) or j5) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 197-215 of SEQ ID NO: 10;
  • j5) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of j4) .
  • the LP primer FIP-Z in the sets of primers provided herein is either of the single-stranded DNA in j7) or j8) as follows:
  • A-linker-B A comprising, consisting essentially of, or consisting of the reverse-complementary sequence of the single-stranded DNA at positions 77-96 of SEQ ID NO: 10; the linker comprising, consisting essentially of, or consisting of a single-stranded DNA of between about 0 and about 10 nucleotides in length; and B comprising, consisting essentially of, or consisting of the sequence of the single-stranded DNA at positions 35-53 of SEQ ID NO: 10;
  • j8) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of j7) .
  • the LP primer BIP-Z in the sets of primers provided herein is either of the single-stranded DNA in j10) or j11) as follows:
  • A-linker-B A comprising, consisting essentially of, or consisting of the sequence of the single-stranded DNA at positions 126-147 of SEQ ID NO: 10; the linker comprising, consisting essentially of, or consisting of a single-stranded DNA of between about 0 and about 10 nucleotides in length; and B comprising, consisting essentially of, or consisting of the reverse-complementary sequence of the single-stranded DNA at positions 179-196 of SEQ ID NO: 10;
  • j11 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of j10) .
  • the LP primer LF-Z in the sets of primers provided herein is either of the single-stranded DNA in j13) or j14) as follows:
  • a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 55-76 of SEQ ID NO: 10;
  • j14 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of j13) .
  • the LP primer LB-Z in the sets of primers provided herein is either of the single-stranded DNA in j16) or j17) as follows:
  • j16 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 154-176 of SEQ ID NO: 10;
  • j17 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of j16) .
  • the LP primer F3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the LP primer F3-Z. In one aspect, the LP primer B3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the LP primer B3-Z. In one aspect, the LP primer FIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the LP primer FIP-Z. In one aspect, the LP primer BIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the LP primer BIP-Z.
  • the LP primer LF-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the LP primer LF-Z. In one aspect, the LP primer LB-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the LP primer LB-Z.
  • the Mycobacterium tuberculosis complex (MTC) primer set can be a primer set comprising, consisting essentially of, or consisting of six sense primers for MTC (e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP) , a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for MTC (e.g., primers comprising the reverse-complementary sequences of the six sense primers) , a primer set comprising, consisting essentially of, or consisting of four sense primers for MTC (e.g., the F3, B3, FIP, and BIP primers for LAMP) , or a primer set comprising, consisting essentially of, or consisting of four anti-sense primers for MTC (e.g., primers comprising the reverse-complementary sequences of the four sense primers) .
  • six sense primers for MTC e.g., the F3, B3, F
  • the MTC primer set comprises MTC primer F3-Z, MTC primer B3-Z, MTC primer FIP-Z, and MTC primer BIP-Z, and optionally MTC primer LF-Z and MTC primer LB-Z, for example, as shown in Table 1.
  • the MTC primer set comprises MTC primer F3-F, MTC primer B3-F, MTC primer FIP-F, and MTC primer BIP-F, and optionally MTC primer LF-F and MTC primer LB-F, for example, as shown in Table 2.
  • the MTC primer F3-Z in the sets of primers provided herein is either of the single-stranded DNA in k1) or k2) as follows:
  • k1 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 14-30 of SEQ ID NO: 11;
  • k2) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of k1) .
  • the MTC primer B3-Z in the sets of primers provided herein is either of the single-stranded DNA in k4) or k5) as follows:
  • k4 a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 175-190 of SEQ ID NO: 11;
  • k5) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of k4) .
  • the MTC primer FIP-Z in the sets of primers provided herein is either of the single-stranded DNA in k7) or k8) as follows:
  • A-linker-B A comprising, consisting essentially of, or consisting of the reverse-complementary sequence of the single-stranded DNA at positions 68-90 of SEQ ID NO: 11; the linker comprising, consisting essentially of, or consisting of a single-stranded DNA of between about 0 and about 10 nucleotides in length; and B comprising, consisting essentially of, or consisting of the sequence of the single-stranded DNA at positions 32-51 of SEQ ID NO: 11;
  • k8 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of k7) .
  • the MTC primer BIP-Z in the sets of primers provided herein is either of the single-stranded DNA in k10) or k11) as follows:
  • A-linker-B A comprising, consisting essentially of, or consisting of the sequence of the single-stranded DNA at positions 113-130 of SEQ ID NO: 11; the linker comprising, consisting essentially of, or consisting of a single-stranded DNA of between about 0 and about 10 nucleotides in length; and B comprising, consisting essentially of, or consisting of the reverse-complementary sequence of the single-stranded DNA at positions 147-167 of SEQ ID NO: 11;
  • k11 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of k10) .
  • the MTC primer LF-Z in the sets of primers provided herein is either of the single-stranded DNA in k13) or k14) as follows:
  • k13 a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 52-67 of SEQ ID NO: 11;
  • k14 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of k13) .
  • the MTC primer LB-Z in the sets of primers provided herein is either of the single-stranded DNA in k16) or k17) as follows:
  • k16 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 131-144 of SEQ ID NO: 11;
  • k17 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of k16) .
  • the MTC primer F3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MTC primer F3-Z. In one aspect, the MTC primer B3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MTC primer B3-Z. In one aspect, the MTC primer FIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MTC primer FIP-Z. In one aspect, the MTC primer BIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MTC primer BIP-Z.
  • the MTC primer LF-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MTC primer LF-Z. In one aspect, the MTC primer LB-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MTC primer LB-Z.
  • the Mycoplasma pneumoniae (MP) primer set can be a primer set comprising, consisting essentially of, or consisting of six sense primers for MP (e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP) , a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for MP (e.g., primers comprising the reverse-complementary sequences of the six sense primers) , a primer set comprising, consisting essentially of, or consisting of four sense primers for MP (e.g., the F3, B3, FIP, and BIP primers for LAMP) , or a primer set comprising, consisting essentially of, or consisting of four anti-sense primers for MP (e.g., primers comprising the reverse-complementary sequences of the four sense primers) .
  • six sense primers for MP e.g., the F3, B3, FIP, BIP, LF, and LB
  • the MP primer set comprises MP primer F3-Z, MP primer B3-Z, MP primer FIP-Z, and MP primer BIP-Z, and optionally MP primer LF-Z and MP primer LB-Z, for example, as shown in Table 1.
  • the MP primer set comprises MP primer F3-F, MP primer B3-F, MP primer FIP-F, and MP primer BIP-F, and optionally MP primer LF-F and MP primer LB-F, for example, as shown in Table 2.
  • the MP primer F3-Z in the sets of primers provided herein is either of the single-stranded DNA in m1) or m2) as follows:
  • m1) a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 14-30 of SEQ ID NO: 12;
  • m2) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of m1) .
  • the MP primer B3-Z in the sets of primers provided herein is either of the single-stranded DNA in m4) or m5) as follows:
  • m4) a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 179-195 of SEQ ID NO: 12;
  • m5) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of m4) .
  • the MP primer FIP-Z in the sets of primers provided herein is either of the single-stranded DNA in m7) or m8) as follows:
  • a single-stranded DNA of the formula A-linker-B comprising, consisting essentially of, or consisting of the reverse-complementary sequence of the single-stranded DNA at positions 71-89 of SEQ ID NO: 12; the linker comprising, consisting essentially of, or consisting of a single-stranded DNA of between about 0 and about 10 nucleotides in length; and B comprising, consisting essentially of, or consisting of the sequence of the single-stranded DNA at positions 31-51 of SEQ ID NO: 12;
  • m8) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of m7) .
  • the MP primer BIP-Z in the sets of primers provided herein is either of the single-stranded DNA in m10) or m11) as follows:
  • A-linker-B A comprising, consisting essentially of, or consisting of the sequence of the single-stranded DNA at positions 99-117 of SEQ ID NO: 12; the linker comprising, consisting essentially of, or consisting of a single-stranded DNA of between about 0 and about 10 nucleotides in length; and B comprising, consisting essentially of, or consisting of the reverse- complementary sequence of the single-stranded DNA at positions 155-171 of SEQ ID NO: 12;
  • m11 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of m10) .
  • the MP primer LF-Z in the sets of primers provided herein is either of the single-stranded DNA in m13) or m14) as follows:
  • m13 a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 52-67 of SEQ ID NO: 12;
  • m14 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of m13) .
  • the MP primer LB-Z in the sets of primers provided herein is either of the single-stranded DNA in m16) or m17) as follows:
  • m16 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 123-138 of SEQ ID NO: 12;
  • m17 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of m16) .
  • the MP primer F3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MP primer F3-Z.
  • the MP primer B3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MP primer B3-Z.
  • the MP primer FIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MP primer FIP-Z.
  • the MP primer BIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MP primer BIP-Z.
  • the MP primer LF-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MP primer LF-Z.
  • the MP primer LB-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the MP primer LB-Z.
  • the Chlamydia pneumoniae (CP) primer set can be a primer set comprising, consisting essentially of, or consisting of six sense primers for CP (e.g., the F3, B3, FIP, BIP, LF, and LB primers for LAMP) , a primer set comprising, consisting essentially of, or consisting of six anti-sense primers for CP (e.g., primers comprising the reverse-complementary sequences of the six sense primers) , a primer set comprising, consisting essentially of, or consisting of four sense primers for CP (e.g., the F3, B3, FIP, and BIP primers for LAMP) , or a primer set comprising, consisting essentially of, or consisting of four anti-sense primers for CP (e.g., primers comprising the reverse-complementary sequences of the four sense primers) .
  • six sense primers for CP e.g., the F3, B3, FIP, BIP
  • the CP primer set comprises CP primer F3-Z, CP primer B3-Z, CP primer FIP-Z, and CP primer BIP-Z, and optionally CP primer LF-Z and CP primer LB-Z, for example, as shown in Table 1.
  • the CP primer set comprises CP primer F3-F, CP primer B3-F, CP primer FIP-F, and CP primer BIP-F, and optionally CP primer LF-F and CP primer LB-F, for example, as shown in Table 2.
  • the CP primer F3-Z in the sets of primers provided herein is either of the single-stranded DNA in n1) or n2) as follows:
  • n1 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 7-30 of SEQ ID NO: 13;
  • n2) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of n1) .
  • the CP primer B3-Z in the sets of primers provided herein is either of the single-stranded DNA in n4) or n5) as follows:
  • n4) a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 203-220 of SEQ ID NO: 13;
  • n5 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of n4) .
  • the CP primer FIP-Z in the sets of primers provided herein is either of the single-stranded DNA in n7) or n8) as follows:
  • A-linker-B A comprising, consisting essentially of, or consisting of the reverse-complementary sequence of the single-stranded DNA at positions 76-95 of SEQ ID NO: 13; the linker comprising, consisting essentially of, or consisting of a single-stranded DNA of between about 0 and about 10 nucleotides in length; and B comprising, consisting essentially of, or consisting of the sequence of the single-stranded DNA at positions 35-54 of SEQ ID NO: 13;
  • n8) a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of n7) .
  • the CP primer BIP-Z in the sets of primers provided herein is either of the single-stranded DNA in n10) or n11) as follows:
  • n11 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of n10) .
  • the CP primer LF-Z in the sets of primers provided herein is either of the single-stranded DNA in n13) or n14) as follows:
  • n13 a single-stranded DNA comprising, consisting essentially of, or consisting of the reverse-complementary sequence of nucleotides at positions 55-75 of SEQ ID NO: 13;
  • n14 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of n13) .
  • the CP primer LB-Z in the sets of primers provided herein is either of the single-stranded DNA in n16) or n17) as follows:
  • n16 a single-stranded DNA comprising, consisting essentially of, or consisting of the sequence of nucleotides at positions 157-179 of SEQ ID NO: 13;
  • n17 a single-stranded DNA of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity with the single-stranded DNA of n16) .
  • the CP primer F3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the CP primer F3-Z.
  • the CP primer B3-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the CP primer B3-Z.
  • the CP primer FIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the CP primer FIP-Z.
  • the CP primer BIP-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the CP primer BIP-Z.
  • the CP primer LF-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the CP primer LF-Z.
  • the CP primer LB-F provided herein comprises, consists essentially of, or consists of the reverse complementary sequence of the CP primer LB-Z.
  • an antisense sequence is a reverse-complement sequence of the sense sequence.
  • the nucleotide sequence of the linker in the SP primer FIP-Z is GTAG. In another aspect, the nucleotide sequence of the linker in the SP primer BIP-Z is AACAAC. In one aspect, the Staphylococcus aureus (SA) is Staphylococcus aureus subsp. (SAS) . In one embodiment, the length of the linker in SAS primer FIP-Z is 0. In one aspect, the length of the linker in SAS primer BIP-Z is 0. In one aspect, the length of the linker in MRSA primer FIP-Z is 0. In one aspect, the length of the linker in MRSA primer BIP-Z is 0. In one aspect, the nucleotide sequence of the linker in the E.
  • the nucleotide sequence of the linker in the E. coli primer BIP-Z is TTT. In one aspect, the length of the linker in the KP primer FIP-Z is 0. In one aspect, the nucleotide sequence of the linker in the KP primer BIP-Z is TTTT. In one aspect, the length of the linker in the PA primer FIP-Z is 0. In one aspect, the length of the linker in the PA primer BIP-Z is 0. In one aspect, the length of the linker in the AB primer FIP-Z is 0. In one aspect, the length of the linker in the AB primer BIP-Z is 0.
  • the length of the linker in the SMA primer FIP-Z is 0. In one aspect, the length of the linker in the SMA primer BIP-Z is 0. In one aspect, the nucleotide sequence of the linker in the HI primer FIP-Z is ATCAAC. In one aspect, the length of the linker in the HI primer BIP-Z is 0. In one aspect, the length of the linker in the LP primer FIP-Z is 0. In one aspect, the length of the linker in the LP primer BIP-Z is 0. In one aspect, the length of the linker in the MTC primer FIP-Z is 0. In one aspect, the length of the linker in the MTC primer BIP-Z is 0.
  • the length of the linker in the MP primer FIP-Z is 0. In one aspect, the length of the linker in the MP primer BIP-Z is 0. In one aspect, the length of the linker in the CP primer FIP-Z is 0. In one aspect, the nucleotide sequence of the linker in the CP primer BIP-Z is TT.
  • the annealing temperature for the LAMP reaction is the temperature at which a primer and a template anneal.
  • each of the primer sets disclosed herein can be individually packaged, for example, as part of a detection kit.
  • Each primer of a primer set can also be individually packaged, and can be combined with other primer (s) at the time of detection.
  • identity and “sequence identity” can refer to a nucleic acid sequence with sequence similarity. In some aspects, “identity” and “sequence identity” refer to nucleic acid sequences having about 85%or more, about 90%or more, or about 95%or more sequence identity. Identity can be evaluated with the naked eye or using a computer software. For example, by using a computer software, identity between two or more sequences can be expressed as a percentage (%) to indicate their identity. A sequence identity of about 85%or more can include about 90%or more, or about 95%or more identity.
  • a primer with an “F” in the name indicates it is an antisense sequence (e.g., antisense DNA sequence) , which is a reverse-complement sequence of a primer with a “Z” in the name.
  • the nucleotide sequence of the CP primer B3-F is a reverse-complement sequence of the CP primer B3-Z.
  • the molar amounts of the primers in the sense primer sets provided herein are:
  • the “F3-Z” primers between about 0.2 ⁇ mol and about 0.3 ⁇ mol (e.g., 0.2 ⁇ mol or 0.3 ⁇ mol) ;
  • the “B3-Z” primers between about 0.2 ⁇ mol and about 0.3 ⁇ mol (e.g., 0.2 ⁇ mol or 0.3 ⁇ mol) ;
  • the “FIP-Z” primers between about 0.8 ⁇ mol and about 2.4 ⁇ mol (e.g., 2.4 ⁇ mol) ;
  • the “BIP-Z” primers between about 0.8 ⁇ mol and about 2.4 ⁇ mol (e.g., 2.4 ⁇ mol) ;
  • the “LF-Z” primers between about 0.4 ⁇ mol and about 1.0 ⁇ mol (e.g., 1.0 ⁇ mol) ;
  • the “LB-Z” primers between about 0.4 ⁇ mol and about 1.0 ⁇ mol (e.g., 1.0 ⁇ mol) .
  • the molar amounts of the primers in the sense primer sets provided herein are:
  • the “F3-Z” primers between about 0.2 ⁇ mol and about 0.3 ⁇ mol (e.g., 0.2 ⁇ mol or 0.3 ⁇ mol) ;
  • the “B3-Z” primers between about 0.2 ⁇ mol and about 0.3 ⁇ mol (e.g., 0.2 ⁇ mol or 0.3 ⁇ mol) ;
  • the “FIP-Z” primers between about 0.8 ⁇ mol and about 2.4 ⁇ mol (e.g., 2.4 ⁇ mol) ;
  • the “BIP-Z” primers between about 0.8 ⁇ mol and about 2.4 ⁇ mol (e.g., 2.4 ⁇ mol) .
  • the molar amounts of the primers in the antisense primer sets provided herein are:
  • the “F3-F” primers between about 0.2 ⁇ mol and about 0.3 ⁇ mol (e.g., 0.2 ⁇ mol or 0.3 ⁇ mol) ;
  • the “B3-F” primers between about 0.2 ⁇ mol and about 0.3 ⁇ mol (e.g., 0.2 ⁇ mol or 0.3 ⁇ mol) ;
  • the “FIP-F” primers between about 0.8 ⁇ mol and about 2.4 ⁇ mol (e.g., 2.4 ⁇ mol) ;
  • the “BIP-F” primers between about 0.8 ⁇ mol and about 2.4 ⁇ mol (e.g., 2.4 ⁇ mol) ;
  • the “LF-F” primers between about 0.4 ⁇ mol and about 1.0 ⁇ mol (e.g., 1.0 ⁇ mol) ;
  • the “LB-F” primers between about 0.4 ⁇ mol and about 1.0 ⁇ mol (e.g., 1.0 ⁇ mol) .
  • the molar amounts of the primers in the antisense primer sets provided herein are:
  • the “F3-F” primers between about 0.2 ⁇ mol and about 0.3 ⁇ mol (e.g., 0.2 ⁇ mol or 0.3 ⁇ mol) ;
  • the “B3-F” primers between about 0.2 ⁇ mol and about 0.3 ⁇ mol (e.g., 0.2 ⁇ mol or 0.3 ⁇ mol) ;
  • the “FIP-F” primers between about 0.8 ⁇ mol and about 2.4 ⁇ mol (e.g., 2.4 ⁇ mol) ;
  • the “BIP-F” primers between about 0.8 ⁇ mol and about 2.4 ⁇ mol (e.g., 2.4 ⁇ mol) .
  • the molar amounts of the primers in the sense primer sets provided herein are: the “F3-Z” primers: 0.3 ⁇ mol; the “B3-Z” primers: 0.3 ⁇ mol; the “FIP-Z” primers: 2.4 ⁇ mol; the “BIP-Z” primers: 2.4 ⁇ mol; the “LF-Z” primers: 1.0 ⁇ mol; and the “LB-Z” primers: 1.0 ⁇ mol.
  • the molar amounts of the primers in the sense primer sets provided herein are: the “F3-Z” primers: 0.3 ⁇ mol; the “B3-Z” primers: 0.3 ⁇ mol; the “FIP-Z” primers: 2.4 ⁇ mol; and the “BIP-Z” primers: 2.4 ⁇ mol.
  • the molar amounts of the primers in the antisense primer sets provided herein are: the “F3-F” primers: 0.3 ⁇ mol; the “B3-F” primers: 0.3 ⁇ mol; the “FIP-F” primers: 2.4 ⁇ mol; the “BIP-F” primers: 2.4 ⁇ mol; the “LF-F” primers: 1.0 ⁇ mol; and the “LB-F” primers: 1.0 ⁇ mol.
  • the molar amounts of the primers in the sense primer sets provided herein are: the “F3-F” primers: 0.3 ⁇ mol; the “B3-F” primers: 0.3 ⁇ mol; the “FIP-F” primers: 2.4 ⁇ mol; and the “BIP-F” primers: 2.4 ⁇ mol.
  • the above-mentioned molar amount is the total number of moles, and the molar ratio is between the total molar amount of each primer. In one aspect, the total number of moles of each primer is the total molar amount of a variety of single-stranded DNAs of each primer.
  • Another technical problem to be solved by the present disclosure is to provide the preparation of LAMP amplification primers for the detection of pathogenic microorganisms related to respiratory tract infections.
  • the present disclosure provides a method for the preparation of LAMP amplification primer (s) for the detection of pathogenic microorganisms related to respiratory tract infections.
  • a method for packaging each LAMP amplification primer or primer set can be individually packaged and/or used to detect the respiratory infections related pathogenic microorganism.
  • the primers and/or primer sets can be mixed and matched for detecting various combinations of the pathogenic microorganisms, for example, simultaneously or sequentially, in the same sample or in the same reaction run of different samples.
  • a chip for detecting respiratory tract infection-related pathogens.
  • the chip comprises a substrate and at least one of the primers and/or primer sets disclosed herein immobilized on the substrate.
  • the chip comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 primer set (s) disclosed herein immobilized on the substrate, and each of the primer sets is specific for detecting a pathogen.
  • the chip is an isothermal amplification microfluidic chip, for example, for performing a LAMP reaction.
  • the system comprises at least one of the primers and/or primer sets disclosed herein, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 primer set (s) , each of the primer sets being specific for detecting a pathogen.
  • the system comprises a chip for the detection of one or more respiratory tract infection related pathogens, such as a microorganism.
  • the system can further comprise one or more reagents and/or one or more instruments and/or one or more data processors (e.g., for analyzing the amplification products) .
  • the one or more reagents do not include the LAMP primers disclosed herein for detecting the pathogen (s) .
  • the amplified product data processor can be used to determine whether a test sample contains one or more pathogen-specific amplification products due to the amplification by the LAMP primers.
  • the LAMP reagent (s) for detecting the respiratory tract infection-related pathogens comprise (s) one or more of: a strand displacement-type DNA polymerase, betaine, dATP, dCTP, dGTP, dTTP, and Mg 2+ .
  • the strand displacement-type DNA polymerase is a Bst DNA polymerase large fragment.
  • the reagent (s) can further comprise a marker, for example, a fluorescent reagent or a coloring reagent, for analyzing one or more amplification products.
  • the fluorescent reagent is EvaGreen.
  • a device or an instrument for use in LAMP for detecting respiratory tract infection pathogens is or comprises an isothermal amplification of nucleic acid microfluidic chip, and optionally an analyzer for the chip.
  • an amplification product data processor is equipped with the analyzer, as are optional software packages.
  • the instrument may be a fluorescence quantitative PCR, and the amplification product data processor can be set within the real-time PCR instrument, and optionally provided with supporting software.
  • the method comprises a step of preparing at least one or all of the LAMP primers for detecting the respiratory tract infection pathogen: a) the primers to detect the respiratory tract infection pathogens; b) the chip to detect the respiratory tract infection pathogens; c) the system to detect the respiratory tract infection pathogens.
  • any of the following applications of LAMP primers to detect the respiratory tract infection pathogenic microorganisms is also provided herein: 1) the application of LAMP primers to detect the respiratory tract infection pathogenic microorganisms used in the preparation of chip to detect the respiratory tract infection pathogenic microorganisms; 2) the application of LAMP primers to detect the respiratory tract infection pathogenic microorganisms used in the preparation of system to detect the respiratory tract infection pathogenic microorganisms; and/or 3) the application of LAMP primers to detect the respiratory tract infection pathogenic microorganisms used in the preparation of a reagent or kit to detect the respiratory tract infection pathogenic microorganisms.
  • a method for detecting the respiratory tract infection pathogenic microorganisms comprising A and/or B below:
  • the respiratory tract infection (RTI) -related pathogenic microorganisms can be the following thirteen sets, any twelve sets, any eleven sets, any ten sets, any nine sets, any eight sets, any seven sets, any six sets, any five sets, any four sets, any three sets, any two sets or any one set: Streptococcus pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Haemophilus influenzae, Legionella pneumophila, Mycobacterium tuberculosis complex, mycoplasma pneumoniae, and Chlamydia pneumonia.
  • Mycobacterium tuberculosis complex refers to a genetically related group of Mycobacterium species that can cause tuberculosis in humans or other organisms.
  • the group includes Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis and the Bacillus Calmette–Guérin strain, Mycobacterium microti, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae, and/or Mycobacterium mungi.
  • the Staphylococcus aureus can be Staphylococcus aureus subsp. aureus (SAS) .
  • test sample can be derived from the respiratory tract of human (Homo sapiens) .
  • the minimum detection limits were 5 ⁇ 10 2 copies/ ⁇ l, for the 13 primer sets provided herein to the Streptococcus pneumoniae DNA, Escherichia coli DNA, Stenotrophomonas maltophilia DNA, Legionella pneumophila DNA, Mycoplasma pneumoniae DNA, Chlamydia pneumoniae DNA, Staphylococcus aureus Golden subspecies DNA, methicillin-resistant Staphylococcus aureus DNA, Klebsiella pneumoniae DNA, Pseudomonas aeruginosa DNA, Acinetobacter baumannii DNA, Haemophilus influenzae DNA and Mycobacterium tuberculosis complex DNA.
  • the thermostatic amplification system is described as follows.
  • the reaction volume for the thermostatic amplification is 20 ⁇ l, comprising 5.5 ⁇ l of 10 ⁇ ThermoPol Buffer, 8.7 ⁇ l of 5M betaine, 0.5 ⁇ l of 50 mg/ml BSA, 0.5 ⁇ l of 400 mM MgSO 4 , 1.6 ⁇ l of 20 ⁇ EvaGreen, 0.2 ⁇ l of 400 mM dNTPs, 2.2 ⁇ l of 8 U/ ⁇ l Bst DNA polymerase large fragment.
  • the primers in the examples were synthesized by Shanghai Sangon Biotech Co., Ltd.
  • the Bst DNA polymerase large fragment and 10 ⁇ ThermoPol Buffer were produced by NEB Company, the betaine was from Sigma Company, and the 20 ⁇ EvaGreen was from Biotium Company.
  • the Proteus vulgaris strain CGMCC No. 1.1527 used in the following examples was deposited in China General Microbiological Culture Collection Center (CGMCC, Address: Institute of Microbiology, Chinese Academy of Sciences (IMCAS) , Court 1, No. 3, Beichen West Road, Chaoyang District, 100101, Beijing, China) on July 1, 1981. This strain can be obtained by the public from the CGMCC.
  • the Proteus vulgaris CGMCC No. 1.1527 strain is hereinafter referred to as PV.
  • the Enterococcus faecium strain CGMCC No. 1.2025 used in the following examples was deposited in China General Microbiological Culture Collection Center (CGMCC, Address: Institute of Microbiology, Chinese Academy of Sciences (IMCAS) , Court 1, No. 3, Beichen West Road, Chaoyang District, 100101, Beijing, China) on August 12, 1996. This strain can be obtained by the public from the CGMCC.
  • the Enterococcus faecium strain CGMCC No. 1.2025 is hereinafter referred to as EF.
  • the Streptococcus mutans strain CGMCC No. 1.2499 used in the following examples was deposited in China General Microbiological Culture Collection Center (CGMCC, Address: Institute of Microbiology, Chinese Academy of Sciences (IMCAS) , Court 1, No. 3, Beichen West Road, Chaoyang District, 100101, Beijing, China) on February 28, 2000. This strain can be obtained by the public from the CGMCC.
  • the Streptococcus mutans strain CGMCC No. 1.2499 is hereinafter referred to as SM.
  • the Citrobacter freundii strain ATCC No. 8090 used in the following examples was deposited in American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA 20110, USA) . This strain can be obtained by the public from the ATCC.
  • the Citrobacter freundii strain ATCC No. 8090 is hereinafter referred to as CF.
  • the Acinetobacter lwoffii strain ATCC No. 15309 used in the following examples was deposited in American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA 20110, USA) . This strain can be obtained by the public from the ATCC.
  • the Acinetobacter lwoffii strain ATCC No. 15309 is hereinafter referred to as AL.
  • the Streptococcus pneumoniae strain ATCC No. 6301 used in the following examples was deposited in American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA 20110, USA) . This strain can be obtained by the public from the ATCC.
  • the Streptococcus pneumoniae strain ATCC No. 6301 is hereinafter referred to as SP.
  • the Escherichia coli strain CGMCC No. 1.8732 used in the following examples was deposited in China General Microbiological Culture Collection Center (CGMCC, Address: Institute of Microbiology, Chinese Academy of Sciences (IMCAS) , Court 1, No. 3, Beichen West Road, Chaoyang District, 100101, Beijing, China) . This strain can be obtained by the public from the CGMCC.
  • the Escherichia coli strain CGMCC No. 1.8732 is hereinafter referred to as E. coli.
  • the Stenotrophomonas maltophilia strain ATCC No. 31559 used in the following examples was deposited in American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA 20110, USA) . This strain can be obtained by the public from the ATCC.
  • the Stenotrophomonas maltophilia strain ATCC No. 31559 is hereinafter referred to as SMA.
  • the Legionella pneumophila strain ATCC No. 33153 used in the following examples was deposited in American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA 20110, USA) . This strain can be obtained by the public from the ATCC.
  • the Legionella pneumophila strain ATCC No. 33153 is hereinafter referred to as LP.
  • the Mycoplasma pneumoniae strain ATCC No. 15531D TM used in the following examples was deposited in American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA 20110, USA) . This strain can be obtained by the public from the ATCC.
  • the Mycoplasma pneumoniae strain ATCC No. 15531D TM is hereinafter referred to as MP.
  • the Chamydia pneumoniae strain ATCC No. VR-2282 TM used in the following examples was deposited in American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA 20110, USA) . This strain can be obtained by the public from the ATCC.
  • the Chamydia pneumoniae strain ATCC No. VR-2282 TM is hereinafter referred to as CP.
  • the Staphylococcus aureus subsp. Aureus strain CGMCC No. 1.8721 used in the following examples was deposited in China General Microbiological Culture Collection Center (CGMCC, Address: Institute of Microbiology, Chinese Academy of Sciences (IMCAS) , Court 1, No. 3, Beichen West Road, Chaoyang District, 100101, Beijing, China) . This strain can be obtained by the public from the CGMCC.
  • the Staphylococcus aureus subsp. Aureus strain CGMCC No. 1.8721 is hereinafter referred to as SAS.
  • the Klebsiella pneumoniae strain ATCC No. 31488 used in the following examples was deposited in American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA 20110, USA) . This strain can be obtained by the public from the ATCC.
  • the Klebsiella pneumoniae strain ATCC No. 31488 is hereinafter referred to as KP.
  • the Pseudomonas aeruginosa strain CGMCC No. 1.2620 in the following examples was reserved in China General Microbiological Culture Collection Center (CGMCC, Address: Institute of Microbiology, Chinese Academy of Sciences (IMCAS) , Court 1, No. 3, Beichen West Road, Chaoyang District, 100101, Beijing, China) on June 6, 2000. This strain can be obtained by the public from the CGMCC.
  • the Pseudomonas aeruginosa strain CGMCC No. 1.2620 is hereinafter referred to as PA.
  • the Acinetobacter baumannii strain ATCC No. BAA-1710 TM used in the following examples was deposited in American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA 20110, USA) . This strain can be obtained by the public from the ATCC.
  • the Acinetobacter baumannii strain ATCC No. BAA-1710 TM is hereinafter referred to as AB.
  • the Haemophilus influenzae ATCC No. 49766 used in the following examples was deposited in American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA 20110, USA) . This strain can be obtained by the public from the ATCC.
  • the Haemophilus influenzae ATCC No. 49766 is hereinafter referred to as HI.
  • the methicillin-resistant Staphylococci (MRS) strain TR558 used in the following examples can be obtained from the applicant. See e.g., Ling-Xiang Zhu et al., “Use of a DNA Microarray for Simultaneous Detection of Antibiotic Resistance Genes among Staphylococcal Clinical Isolates, ” J. Clin. Microbiol. (2007) , 45 (11) : 3514-21, the content of which is incorporated herein by reference in its entirety.
  • the Mycobacterium tuberculosis complex H37Rv used in the following examples can be obtained from the applicant. See e.g., Yang Liu et al., “Application of Hyperbranched Rolling Circle Amplification for Direct Detection of Mycobacterium Tuberculosis in Clinical Sputum Specimens, ” PLoS One (2013) , 8 (6) : e64583. doi: 10.1371/journal. pone. 0064583, the content of which is incorporated herein by reference in its entirety.
  • microfluidic nucleic acid analyzer in the following examples was made by Capitalbio Corporation, catalog number 170011.
  • microfluidic chip in the following examples was made according to the examples of Chinese Patent No. 201420103225.5, filed March 7, 2014, published as CN 203750554 U on August 6, 2014, the content of which is incorporated herein by reference in its entirety.
  • the LAMP primer sets for detection of respiratory tract infection (RTI) -related pathogenic microorganisms include the following thirteen sets, each of which can be individually packaged: SP (Streptococcus pneumoniae) , SA (Staphylococcus aureus) , MRSA (methicillin-resistant Staphylococcus aureus) , E.
  • the underlined nucleotide sequences are linker sequences.
  • the underlined nucleotide sequences are linker sequences.
  • Molar amount of the primers in Table 1 is: “F3-Z” 0.3 ⁇ mol; “B3-Z” 0.3 ⁇ mol; “FIP-Z” 2.4 ⁇ mol; “BIP-Z” 2.4 ⁇ mol; “LF-Z” 1.0 ⁇ mol; “LB-Z” 1.0 ⁇ mol.
  • This example shows the detection of one or more pathogen nucleic acid sequences in a mixture of DNA samples using an isothermal-amplified micro-fluidic chip (IAMFC) .
  • IAMFC isothermal-amplified micro-fluidic chip
  • each primer in the primer set used in Example 1 for detecting SP was embedded with an agarose solution and dried and fixed in reaction tank A1 of the isothermal-amplified micro-fluidic chip.
  • each primer in the primer sets for detecting the SA subspecies (SAS) , MRSA, E. coli, KP, PA, AB, SMA, HI, LP, MTC, MP, and CP were fixed in reaction tanks B1, C1, D1, E1, F1, G1, H1, I1, J1, K1, L1, and M1 of the IAMFC respectively.
  • No primers were fixed in reaction tanks N1, O1, P1, Q1, R1, S1, T1, U1, V1, W1, and X1 of the IAMFC, which were taken as blank controls.
  • the SP DNA was extracted using the bacterial genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd. ) , and the procedures were operated according to the instructions in the DNA extraction kit to obtain the SP DNA. According to the above method, SP was replaced with E. coli, SMA, LP, MP, and CP, with other steps unchanged, to extract the E. coli DNA, SMA DNA, LP DNA, MP DNA, and CP DNA, respectively.
  • the SP DNA, E. coli DNA, SMA DNA, LP DNA, MP DNA, and CP DNA described above were mixed to obtain a mixture system No. 1, in which the concentrations of SP DNA, E. coli DNA, SMA DNA, LP DNA, MP DNA, and CP DNA were 2 ⁇ 10 3 copies/ ⁇ l, respectively.
  • the DNA in the mixture system No. 1 was named as mixture DNA No. 1.
  • the DNA No. 1 was mixed with the isothermal amplification system, and then injected into the IAMFC in step 2.1 for isothermal amplification reactions and data analysis according to the operating instructions in the IAMFC nucleic acid analyzer.
  • the reaction temperature was 65°C and the reaction time was 50 minutes.
  • the amplification curves corresponding to reaction tanks A1, D1, H1, J1, L1, and M1 (which contained fixed primer sets for SP, E. coli, SMA, LP, MP, and CP, respectively) were the typical “S-shape” amplification curves.
  • the amplification curves corresponding to reaction tanks B1, C1, E1, F1, G1, I1, and K1 (which contained fixed primer sets for SAS, MRSA, KP, PA, AB, HI, and MTC, respectively) were horizontal straight lines, the same as reaction tanks N1 to X1 in which no primer sets were fixed.
  • each primer in primer sets for detecting SP, SAS, MRSA, E. coli, KP, PA, AB, SMA, HI, LP, MTC, MP, and CP was fixed in reaction tanks A2, B2, C2, D2, E2, F2, G2, H2, I2, J2, K2, L2, and M2 of the IAMFC, respectively; the reaction tanks N2, O2, P2, Q2, R2, S2, T2, U2, V2, W2, and X2 of the IAMFC did not contain the primer sets and were taken as blank controls.
  • the SA DNA was extracted using the bacterial genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd. ) , and the procedures were operated according to the instructions in the DNA extraction kit to obtain the SAS DNA.
  • SAS was replaced with MRSA, KP, PA, AB, HI, and MTC, with other steps unchanged, to extract the MRSA DNA, KP DNA, PA DNA, ABDNA, HI DNA, and MTC DNA respectively.
  • the SAS DNA, MRSA DNA, KP DNA, PA DNA, AB DNA, HI DNA, and MTC DNA described above were mixed to obtain a mixture system No. 2, in which the concentrations of SAS DNA, MRSA DNA, KP DNA, PA DNA, AB DNA, HI DNA, and MTC DNA were 2 ⁇ 10 3 copies/ ⁇ l respectively.
  • the DNA in the mixture system No. 2 was named as mixture DNA No. 2.
  • the DNA No. 2 was mixed with the isothermal amplification system, and then injected into the IAMFC in step 3.1 for isothermal amplification reactions and data analysis according to the operating instructions in the IAMFC nucleic acid analyzer.
  • the reaction temperature was 65°C and the reaction time was 50 minutes.
  • the amplification curves corresponding to reaction tanks A2, D2, H2, J2, L2, and M2 (which contained fixed primer sets for SP, E. coli, SMA, LP, MP, and CP, respectively) were horizontal straight lines.
  • the amplification curves corresponding to reaction tanks B2, C2, E2, F2, G2, I2, and K2 (which contained fixed primer sets for SAS, MRSA, KP, PA, AB, HI and MTC, respectively) were the typical “S-shape” amplification curves.
  • the amplification curves corresponding to reaction tanks N2 to X2 (in which no primer sets were fixed) were also horizontal straight lines. The results indicated that the mixture DNA No.
  • each primer in the primer sets for detecting SP, SAS, MRSA, E. coli, KP, PA, AB, SMA, HI, LP, MTC, MP, and CP was fixed in reaction tanks A3, B3, C3, D3, E3, F3, G3, H3, I3, J3, K3, L3 and M3 of the IAMFC respectively; the reaction tank N3, O3, P3, Q3, R3, S3, T3, U3, V3, W3, and X3 of the IAMFC did not contain any fixed primers and were taken as blank controls.
  • the nucleic acid of clinical sputum sample from a pneumonia patient was extracted using the nucleic acid extraction reagent (CapitalBio Technology) , mixed with the isothermal amplification system, and then injected into the IAMFC in step 4.1 for isothermal amplification reactions and data analysis according to the operating instructions in the IAMFC nucleic acid analyzer.
  • the reaction temperature was 65°C and the reaction time was 50 minutes.
  • the amplification curves corresponding to reaction tanks B3, C3, G3 of primer sets (which contained fixed primer sets for SAS, MRSA and AB, respectively) were the typical “S-shape” amplification curves.
  • the amplification curves corresponding to reaction tanks A3, D3, E3, F3, H3, I3, J3, K3, L3, and M3 (which contained fixed primer sets for SP, E. coli, KP, PA, SMA, HI, LP, MTC, MP, and CP) were horizontal straight lines, the same as reaction tanks N3 to X3 (which contained no fixed primer sets) .
  • the PV DNA was extracted using the bacterial genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd. ) , and the procedures were operated according to the instructions in the DNA extraction kit to obtain the PV DNA.
  • PV was replaced with enterococcus faecium (EF) , streptococcus mutans (SM) , citrbacter freundii (CF) and acinetobacter lwoffii (AL) , with other steps unchanged, to extract the EF DNA, SM DNA, CF DNA, and AL DNA, respectively.
  • EF enterococcus faecium
  • SM streptococcus mutans
  • CF citrbacter freundii
  • AL acinetobacter lwoffii
  • the PV DNA was mixed with the isothermal amplification system, and then injected into the IAMFC in step 5.1 for isothermal amplification reactions and data analysis according to the operating instructions in the IAMFC nucleic acid analyzer.
  • the reaction temperature was 65°C and the reaction time was 50 minutes.
  • PV DNA was replaced with EF DNA, SM DNA, CF DNA, AL DNA, SP DNA, E. coli DNA, SMA DNA, LP DNA, MP DNA, CP DNA, SAS DNA, MRSA DNA , KP DNA, PA DNA, AB DNA, HI DNA, and MTC DNA, with other steps unchanged, to conduct the corresponding data analysis respectively.
  • the amplification curve corresponding to the reaction tank is a typical “S-shape” amplification curve, then the results is positive, indicating presence and detection of the pathogen DNA. If the amplification curve corresponding to the reaction tank is a horizontal straight line, then the result is negative, indicating no presence and no detection of the pathogen DNA.
  • the testing results were shown in Table 3. The results indicated that the present method can specifically detect SP, E. coli, SMA, LP, MP, CP, SAS, MRSA, KP, PA, AB, HI, and MTC, without cross-reaction with other pathogenic microorganisms, such as PV, EF, SM, CF, and AL.
  • the SP DNA, E. coli DNA, SMA DNA, LP DNA, MP DNA, CP DNA, golden yellow SA subspecies DNA (SAS DNA) , MRSA DNA, KP DNA, PA DNA , AB DNA, HI DNA, and MTC DNA prepared in 2.2 of Example 2 and 3.2 of Example 3 were diluted respectively and mixed to obtain the mixture system No. 3, in which the concentrations of each of SP DNA, E. coli DNA, SMA DNA, LP DNA, MP DNA, CP DNA, SAS DNA, MRSA DNA, KP DNA, PA DNA, AB DNA, HI DNA, and MTC DNA was 5 ⁇ 10 2 copies/ ⁇ l.
  • the DNA in the mixture system No. 3 was named as mixture DNA No. 3.
  • the DNA No. 3 was mixed with the isothermal amplification system, and then injected into the IAMFC in step 6.1 for isothermal amplification reactions and data analysis according to the operating instructions in the IAMFC nucleic acid analyzer.
  • the reaction temperature was 65°C and the reaction time was 50 minutes.
  • the testing results showed that the amplification curves corresponding to reaction tanks fixed with primer sets for SP, SAS, MRSA, E. coli, KP, PA, AB, SMA, HI, LP, MTC, MP and CP were the typical S-shaped amplification curves.
  • the amplification curves corresponding to reaction tanks not fixed with any primer sets were horizontal straight lines. Therefore, in one aspect, the minimum detection limits of SP DNA, E. coli DNA, SMA DNA, LP DNA, MP DNA, CP DNA, SAS DNA, MRSA DNA, KP DNA, PA DNA, AB DNA, HI DNA, and MTC DNA were at least all 5 ⁇ 10 2 copies/ ⁇ l.
  • Embodiment 1 A primer set for loop-mediated isothermal amplification (LAMP) for detection of respiratory tract infection (RTI) -related pathogenic microorganisms, which comprises the thirteen primer sets, any twelve sets, any eleven sets, any ten sets, any nine sets, any eight sets, any seven sets, any six sets, any five sets, any four sets, any three sets, any two sets, or any one set selected from the group consisting of:
  • a primer set for detecting Streptococcus pneumoniae SP
  • a primer set for detecting Staphylococcus aureus SA
  • a primer set for detecting methicillin-resistant Staphylococcus aureus MRSA
  • coli a primer set for detecting Klebsiella pneumoniae (KP) , a primer set for detecting Pseudomonas aeruginosa (PA) , a primer set for detecting Acinetobacter baumannii (AB) , a primer set for detecting Stenotrophomonas maltophilia (SMA) , a primer set for detecting Haemophilus influenzae (HI) , a primer set for detecting Legionella pneumophila (LP) , a primer set for detecting Mycobacterium tuberculosis complex (MTC) , a primer set for detecting Mycoplasma pneumoniae (MP) , and a primer set for detecting Chlamydia pneumoniae (CP) ,
  • KP Klebsiella pneumoniae
  • PA Pseudomonas aeruginosa
  • AB Acinetobacter baumannii
  • SMA Stenotrophomonas maltophilia
  • the primer set for detecting Streptococcus pneumoniae comprises six sense primers: SP primer F3-Z, SP primer B3-Z, SP primer FIP-Z, SP primer BIP-Z, SP primer LF-Z, and SP primer LB-Z; six antisense primers: SP primer F3-F, SP primer B3-F, SP primer FIP-F, SP primer BIP-F, SP primer LF-F, and SP primer LB-F; four sense primers: SP primer F3-Z, SP primer B3-Z, SP primer FIP-Z, and SP primer BIP-Z; or four antisense primers: SP primer F3-F, SP primer B3-F, SP primer FIP-F, and SP primer BIP-F,
  • SP primer F3-Z comprises either the single-stranded DNA in a1) or a2) as follows:
  • SP primer B3-Z comprises either the single-stranded DNA in a4) or a5) as follows:
  • SP primer FIP-Z comprises either the single-stranded DNA in a7) or a8) as follows:
  • A-linker-B a single-stranded DNA with a structure of A-linker-B, wherein A is a reverse-complement single-stranded DNA of the single-stranded DNA at positions 79-100 of SEQ ID NO: 1, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a single-stranded DNA at positions 39-55 of SEQ ID NO: 1;
  • SP primer BIP-Z comprises either the single-stranded DNA in a10) or a11) as follows:
  • A is a single-stranded DNA at positions 101-124 of SEQ ID NO: 1
  • the linker is a single-stranded DNA with a length of 0-10 nucleotides
  • B is a reverse-complement single-stranded DNA of the single-stranded DNA at position 166-185 of SEQ ID NO: 1;
  • SP primer LF-Z comprises either the single-stranded DNA in a13) or a14) as follows:
  • SP primer LB-Z comprises either the single-stranded DNA in a16) or a17) as follows:
  • the SP primer F3-F comprises or is the antisense DNA of the SP primer F3-Z
  • the SP primer B3-F comprises or is the antisense DNA of the SP primer B3-Z
  • the SP primer FIP-F comprises or is the antisense DNA of the SP primer FIP-Z
  • the SP primer BIP-F comprises or is the antisense DNA of the SP primer BIP-Z
  • the SP primer LF-F comprises or is the antisense DNA of the SP primer LF-Z
  • the SP primer LB-F comprises or is the antisense DNA of the SP primer LB-Z
  • the primer set for detecting Staphylococcus aureus comprises six sense primers: SA primer F3-Z, SA primer B3-Z, SA primer FIP-Z, SA primer BIP-Z, SA primer LF-Z, and SA primer LB-Z; six antisense primers: SA primer F3-F, SA primer B3-F, SA primer FIP-F, SA primer BIP-F, SA primer LF-F, and SA primer LB-F; four sense primers: SA primer F3-Z, SA primer B3-Z, SA primer FIP-Z, and SA primer BIP-Z; or four antisense primers: SA primer F3-F, SA primer B3-F, SA primer FIP-F, and SA primer BIP-F,
  • SA primer F3-Z comprises either the single-stranded DNA in b1) or b2) as follows:
  • SA primer B3-Z comprises either the single-stranded DNA in b4) or b5) as follows:
  • SA primer FIP-Z comprises either the single-stranded DNA in b7) or b8) as follows:
  • A-linker-B a single-stranded DNA with a structure of A-linker-B, wherein A is a reverse-complement single-stranded DNA of the single-stranded DNA of SEQ ID NO: 2 at positions 72-95, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a single-stranded DNA of the sequence at positions 31-50 of SEQ ID NO: 2;
  • SA primer BIP-Z comprises either the single-stranded DNA in b10) or b11) as follows:
  • A-linker-B a single-stranded DNA with a structure of A-linker-B, wherein A is a single-stranded DNA of SEQ ID NO: 2 at positions 127-149, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a reverse-complement single-stranded DNA of the single-stranded DNA at positions 196-212 of SEQ ID NO: 2;
  • SA primer LF-Z comprises either the single-stranded DNA in b13) or b14) as follows:
  • SA primer LB-Z comprises either the single-stranded DNA in b16) or b17) as follows:
  • the SA primer F3-F comprises or is the antisense DNA of the SA primer F3-Z
  • the SA primer B3-F comprises or is the antisense DNA of the SA primer B3-Z
  • the SA primer FIP-F comprises or is the antisense DNA of the SA primer FIP-Z
  • the SA primer BIP-F comprises or is the antisense DNA of the SA primer BIP-Z
  • the SA primer LF-F comprises or is the antisense DNA of the SA primer LF-Z
  • the SA primer LB-F comprises or is the antisense DNA of the SA primer LB-Z
  • the primer set for detecting methicillin-resistant Staphylococcus aureus comprises six sense primers: MRSA primer F3-Z, MRSA primer B3-Z, MRSA primer FIP-Z, MRSA primer BIP-Z, MRSA primer LF-Z, and MRSA primer LB-Z; six antisense primers: MRSA primer F3-F, MRSA primer B3-F, MRSA primer FIP-F, MRSA primer BIP-F, MRSA primer LF-F, and MRSA primer LB-F; four sense primers: MRSA primer F3-Z, MRSA primer B3-Z, MRSA primer FIP-Z, and MRSA primer BIP-Z; or four antisense primers: MRSA primer F3-F, MRSA primer B3-F, MRSA primer FIP-F, and MRSA primer BIP-F,
  • MRSA primer F3-Z comprises either the single-stranded DNA in c1) or c2) as follows:
  • MRSA primer B3-Z comprises either the single-stranded DNA in c4) or c5) as follows:
  • MRSA primer FIP-Z comprises either the single-stranded DNA in c7) or c8) as follows:
  • A-linker-B wherein A is a reverse-complement single-stranded DNA of the single-stranded DNA of SEQ ID NO: 3 at positions 97-121, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a single-stranded DNA of the sequence at positions 47-71 of SEQ ID NO: 3;
  • MRSA primer BIP-Z comprises either the single-stranded DNA in c10) or c11) as follows:
  • B is a reverse-complement single-stranded DNA of the single-stranded DNA at positions 182-206 of SEQ ID NO: 3;
  • MRSA primer LF-Z comprises either the single-stranded DNA in c13) or c14) as follows:
  • MRSA primer LB-Z comprises either the single-stranded DNA in c16) or c17) as follows:
  • the MRSA primer F3-F comprises or is the antisense DNA of the MRSA primer F3-Z
  • the MRSA primer B3-F comprises or is the antisense DNA of the MRSA primer B3-Z
  • the MRSA primer FIP-F comprises or is the antisense DNA of the MRSA primer FIP-Z
  • the MRSA primer BIP-F comprises or is the antisense DNA of the MRSA primer BIP-Z
  • the MRSA primer LF-F comprises or is the antisense DNA of the MRSA primer LF-Z
  • the MRSA primer LB-F comprises or is the antisense DNA of the MRSA primer LB-Z
  • the primer set for detecting E. coli comprises six sense primers: E. coli primer F3-Z, E. coli primer B3-Z, E. coli primer FIP-Z, E. coli primer BIP-Z, E. coli primer LF-Z, and E. coli primer LB-Z; six antisense primers: E. coli primer F3-F, E. coli primer B3-F, E. coli primer FIP-F, E. coli primer BIP-F, E. coli primer LF-F, and E. coli primer LB-F; four sense primers: E. coli primer F3-Z, E. coli primer B3-Z, E. coli primer FIP-Z, and E. coli primer BIP-Z; or four antisense primers: E. coli primer F3-F, E. coli primer B3-F, E. coli primer FIP-F, and E. coli primer BIP-F,
  • E. coli primer F3-Z comprises either the single-stranded DNA in d1) or d2) as follows:
  • d2) a single-stranded DNA with over 85%of identity with the single-stranded DNA of d1) ,
  • E. coli primer B3-Z comprises either the single-stranded DNA in d4) or d5) as follows:
  • E. coli primer FIP-Z comprises either the single-stranded DNA in d7) or d8) as follows:
  • A-linker-B wherein A is a reverse-complement single-stranded DNA of the single-stranded DNA of SEQ ID NO: 4 at positions 77-96, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a single-stranded DNA of the sequence at positions 38-55 of SEQ ID NO: 4;
  • E. coli primer BIP-Z comprises either the single-stranded DNA in d10) or d11) as follows:
  • d10) a single-stranded DNA with a structure of A-linker-B, wherein A is a single-stranded DNA of SEQ ID NO: 4 at positions 101-122, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a reverse-complement single-stranded DNA of the single-stranded DNA at positions 163-183 of SEQ ID NO: 4;
  • E. coli primer LF-Z comprises either the single-stranded DNA in d13) or d14) as follows:
  • E. coli primer LB-Z comprises either the single-stranded DNA in d16) or d17) as follows:
  • the E. coli primer F3-F comprises or is the antisense DNA of the E. coli primer F3-Z
  • the E. coli primer B3-F comprises or is the antisense DNA of the E. coli primer B3-Z
  • the E. coli primer FIP-F comprises or is the antisense DNA of the E. coli primer FIP-Z
  • the E. coli primer BIP-F comprises or is the antisense DNA of the E. coli primer BIP-Z
  • the E. coli primer LF-F comprises or is the antisense DNA of the E. coli primer LF-Z
  • the E. coli primer LB-F comprises or is the antisense DNA of the E. coli primer LB-Z
  • the primer set for detecting KP comprises six sense primers: KP primer F3-Z, KP primer B3-Z, KP primer FIP-Z, KP primer BIP-Z, KP primer LF-Z, and KP primer LB-Z; six antisense primers: KP primer F3-F, KP primer B3-F, KP primer FIP-F, KP primer BIP-F, KP primer LF-F, and KP primer LB-F; four sense primers: KP primer F3-Z, KP primer B3-Z, KP primer FIP-Z, and KP primer BIP-Z; or four antisense primers: KP primer F3-F, KP primer B3-F, KP primer FIP-F, and KP primer BIP-F,
  • KP primer F3-Z comprises either of:
  • e1 a single-stranded DNA of nucleotide of SEQ ID NO: 5 at positions 14-30;
  • e2) a single-stranded DNA with over 85%of identity with the single-stranded DNA of e1) ,
  • KP primer B3-Z comprises either of:
  • KP primer FIP-Z comprises either of:
  • the linker is a single-stranded DNA with a length of 0-10 nucleotides
  • B is a single-stranded DNA of the sequence at positions 38-54 of SEQ ID NO: 5;
  • KP primer BIP-Z comprises either of:
  • A-linker-B wherein A is a single-stranded DNA of SEQ ID NO: 5 at positions 118-140, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a reverse-complement single-stranded DNA of the single-stranded DNA at positions 168-185 of SEQ ID NO: 5;
  • KP primer LF-Z comprises either of:
  • KP primer LB-Z comprises either of:
  • the KP primer F3-F comprises or is the antisense DNA of the KP primer F3-Z
  • the KP primer B3-F comprises or is the antisense DNA of the KP primer B3-Z
  • the KP primer FIP-F comprises or is the antisense DNA of the KP primer FIP-Z
  • the KP primer BIP-F comprises or is the antisense DNA of the KP primer BIP-Z
  • the KP primer LF-F comprises or is the antisense DNA of the KP primer LF-Z
  • the KP primer LB-F comprises or is the antisense DNA of the KP primer LB-Z
  • the primer set for detecting PA comprises six sense primers: PA primer F3-Z, PA primer B3-Z, PA primer FIP-Z, PA primer BIP-Z, PA primer LF-Z, and PA primer LB-Z; six antisense primers: PA primer F3-F, PA primer B3-F, PA primer FIP-F, PA primer BIP-F, PA primer LF-F, and PA primer LB-F; four sense primers: PA primer F3-Z, PA primer B3-Z, PA primer FIP-Z, and PA primer BIP-Z; or four antisense primers: PA primer F3-F, PA primer B3-F, PA primer FIP-F, and PA primer BIP-F,
  • PA primer F3-Z comprises either of:
  • PA primer B3-Z comprises either of:
  • PA primer FIP-Z comprises either of:
  • A-linker-B a single-stranded DNA with a structure of A-linker-B, wherein A is a reverse-complement single-stranded DNA of the single-stranded DNA of SEQ ID NO: 6 at positions 75-93, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a single-stranded DNA of the sequence at positions 35-53 of SEQ ID NO: 6;
  • PA primer BIP-Z comprises either of:
  • A-linker-B a single-stranded DNA with a structure of A-linker-B, wherein A is a single-stranded DNA of SEQ ID NO: 6 at positions 126-144, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a reverse-complement single-stranded DNA of the single-stranded DNA at positions 188-205 of SEQ ID NO: 6;
  • PA primer LF-Z comprises either of:
  • PA primer LB-Z comprises either of:
  • the PA primer F3-F comprises or is the antisense DNA of the PA primer F3-Z
  • the PA primer B3-F comprises or is the antisense DNA of the PA primer B3-Z
  • the PA primer FIP-F comprises or is the antisense DNA of the PA primer FIP-Z
  • the PA primer BIP-F comprises or is the antisense DNA of the PA primer BIP-Z
  • the PA primer LF-F comprises or is the antisense DNA of the PA primer LF-Z
  • the PA primer LB-F comprises or is the antisense DNA of the PA primer LB-Z
  • the primer set for detecting AB comprises six sense primers: AB primer F3-Z, AB primer B3-Z, AB primer FIP-Z, AB primer BIP-Z, AB primer LF-Z, and AB primer LB-Z; six antisense primers: AB primer F3-F, AB primer B3-F, AB primer FIP-F, AB primer BIP-F, AB primer LF-F, and AB primer LB-F; four sense primers: AB primer F3-Z, AB primer B3-Z, AB primer FIP-Z, and AB primer BIP-Z; or four antisense primers: AB primer F3-F, AB primer B3-F, AB primer FIP-F, and AB primer BIP-F,
  • AB primer F3-Z comprises either of:
  • g1 a single-stranded DNA of nucleotide of SEQ ID NO: 7 at positions 10-30;
  • g2) a single-stranded DNA with over 85%of identity with the single-stranded DNA of g1) ,
  • AB primer B3-Z comprises either of:
  • AB primer FIP-Z comprises either of:
  • g7 a single-stranded DNA with a structure of A-linker-B, wherein A is a reverse-complement single-stranded DNA of the single-stranded DNA of SEQ ID NO: 7 at positions 73-94, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a single-stranded DNA of the sequence at positions 31-55 of SEQ ID NO: 7;
  • AB primer BIP-Z comprises either of:
  • g10) a single-stranded DNA with a structure of A-linker-B, wherein A is a single-stranded DNA of SEQ ID NO: 7 at positions 128-152, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a reverse-complement single-stranded DNA of the single-stranded DNA at positions 186-204 of SEQ ID NO: 7;
  • g11 a single-stranded DNA with over 85%of identity with the single-stranded DNA of g10) ,
  • AB primer LF-Z comprises either of:
  • AB primer LB-Z comprises either of:
  • g17 a single-stranded DNA fragment with over 85%of identity with the single-stranded DNA of g16) ,
  • the AB primer F3-F comprises or is the antisense DNA of the AB primer F3-Z
  • the AB primer B3-F comprises or is the antisense DNA of the AB primer B3-Z
  • the AB primer FIP-F comprises or is the antisense DNA of the AB primer FIP-Z
  • the AB primer BIP-F comprises or is the antisense DNA of the AB primer BIP-Z
  • the AB primer LF-F comprises or is the antisense DNA of the AB primer LF-Z
  • the AB primer LB-F comprises or is the antisense DNA of the AB primer LB-Z
  • the primer set for detecting SMA comprises six sense primers: SMA primer F3-Z, SMA primer B3-Z, SMA primer FIP-Z, SMA primer BIP-Z, SMA primer LF-Z, and SMA primer LB-Z; six antisense primers: SMA primer F3-F, SMA primer B3-F, SMA primer FIP-F, SMA primer BIP-F, SMA primer LF-F, and SMA primer LB-F; four sense primers: SMA primer F3-Z, SMA primer B3-Z, SMA primer FIP-Z, and SMA primer BIP-Z; or four antisense primers: SMA primer F3-F, SMA primer B3-F, SMA primer FIP-F, and SMA primer BIP-F,
  • SMA primer F3-Z comprises either of:
  • h1 a single-stranded DNA of nucleotide of SEQ ID NO: 8 at positions 11-30;
  • h2 a single-stranded DNA with over 85%of identity with the single-stranded DNA of h1) ,
  • SMA primer B3-Z comprises either of:
  • SMA primer FIP-Z comprises either of:
  • A-linker-B wherein A is a reverse-complement single-stranded DNA of the single-stranded DNA of SEQ ID NO: 8 at positions 81-100, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a single-stranded DNA of the sequence at positions 41-57 ofSEQ ID NO: 8;
  • SMA primer BIP-Z comprises either of:
  • h10) a single-stranded DNA with a structure of A-linker-B, wherein A is a single-stranded DNA of SEQ ID NO: 8 at positions 109-132, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a reverse-complement single-stranded DNA of the single-stranded DNA at positions 175-193 of SEQ ID NO: 8;
  • SMA primer LF-Z comprises either of:
  • SMA primer LB-Z comprises either of:
  • the SMA primer F3-F comprises or is the antisense DNA of the SMA primer F3-Z
  • the SMA primer B3-F comprises or is the antisense DNA of the SMA primer B3-Z
  • the SMA primer FIP-F comprises or is the antisense DNA of the SMA primer FIP-Z
  • the SMA primer BIP-F comprises or is the antisense DNA of the SMA primer BIP-Z
  • the SMA primer LF-F comprises or is the antisense DNA of the SMA primer LF-Z
  • the SMA primer LB-F comprises or is the antisense DNA of the SMA primer LB-Z
  • the primer set for detecting HI comprises six sense primers: HI primer F3-Z, HI primer B3-Z, HI primer FIP-Z, HI primer BIP-Z, HI primer LF-Z, and HI primer LB-Z; six antisense primers: HI primer F3-F, HI primer B3-F, HI primer FIP-F, HI primer BIP-F, HI primer LF-F, and HI primer LB-F; four sense primers: HI primer F3-Z, HI primer B3-Z, HI primer FIP-Z, and HI primer BIP-Z; or four antisense primers: HI primer F3-F, HI primer B3-F, HI primer FIP-F, and HI primer BIP-F,
  • HI primer F3-Z comprises either of:
  • HI primer B3-Z comprises either of:
  • HI primer FIP-Z comprises either of:
  • A-linker-B wherein A is a reverse-complement single-stranded DNA of the single-stranded DNA of SEQ ID NO: 9 at positions 83-107, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a single-stranded DNA of the sequence at positions 39-58 of SEQ ID NO: 9;
  • HI primer BIP-Z comprises either of:
  • A-linker-B a single-stranded DNA with a structure of A-linker-B, wherein A is a single-stranded DNA of SEQ ID NO: 9 at positions 114-139, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a reverse-complement single-stranded DNA of the single-stranded DNA at positions 176-199 of SEQ ID NO: 9;
  • HI primer LF-Z comprises either of:
  • HI primer LB-Z comprises either of:
  • the HI primer F3-F comprises or is the antisense DNA of the HI primer F3-Z
  • the HI primer B3-F comprises or is the antisense DNA of the HI primer B3-Z
  • the HI primer FIP-F comprises or is the antisense DNA of the HI primer FIP-Z
  • the HI primer BIP-F comprises or is the antisense DNA of the HI primer BIP-Z
  • the HI primer LF-F comprises or is the antisense DNA of the HI primer LF-Z
  • the HI primer LB-F comprises or is the antisense DNA of the HI primer LB-Z
  • the primer set for detecting LP comprises six sense primers: LP primer F3-Z, LP primer B3-Z, LP primer FIP-Z, LP primer BIP-Z, LP primer LF-Z, and LP primer LB-Z; six antisense primers: LP primer F3-F, LP primer B3-F, LP primer FIP-F, LP primer BIP-F, LP primer LF-F, and LP primer LB-F; four sense primers: LP primer F3-Z, LP primer B3-Z, LP primer FIP-Z, and LP primer BIP-Z; or four antisense primers: LP primer F3-F, LP primer B3-F, LP primer FIP-F, and LP primer BIP-F, wherein the LP primer F3-Z comprises either of:
  • LP primer B3-Z comprises either of:
  • LP primer FIP-Z comprises either of:
  • A-linker-B wherein A is a reverse-complement single-stranded DNA of the single-stranded DNA of SEQ ID NO: 10 at positions 77-96, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a single-stranded DNA of the sequence at positions 35-53 of SEQ ID NO: 10;
  • LP primer BIP-Z comprises either of:
  • A-linker-B wherein A is a single-stranded DNA of SEQ ID NO: 10 at positions 126-147, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a reverse-complement single-stranded DNA of the single-stranded DNA at positions 179-196 of SEQ ID NO: 10;
  • LP primer LF-Z comprises either of:
  • LP primer LB-Z comprises either of:
  • the LP primer F3-F comprises or is the antisense DNA of the LP primer F3-Z
  • the LP primer B3-F comprises or is the antisense DNA of the LP primer B3-Z
  • the LP primer FIP-F comprises or is the antisense DNA of the LP primer FIP-Z
  • the LP primer BIP-F comprises or is the antisense DNA of the LP primer BIP-Z
  • the LP primer LF-F comprises or is the antisense DNA of the LP primer LF-Z
  • the LP primer LB-F comprises or is the antisense DNA of the LP primer LB-Z
  • the primer set for detecting MTC comprises six sense primers: MTC primer F3-Z, MTC primer B3-Z, MTC primer FIP-Z, MTC primer BIP-Z, MTC primer LF-Z, and MTC primer LB-Z; six antisense primers: MTC primer F3-F, MTC primer B3-F, MTC primer FIP-F, MTC primer BIP-F, MTC primer LF-F, and MTC primer LB-F; four sense primers: MTC primer F3-Z, MTC primer B3-Z, MTC primer FIP-Z, and MTC primer BIP-Z; or four antisense primers: MTC primer F3-F, MTC primer B3-F, MTC primer FIP-F, and MTC primer BIP-F,
  • MTC primer F3-Z comprises either of:
  • MTC primer B3-Z comprises either of:
  • MTC primer FIP-Z comprises either of:
  • A-linker-B a single-stranded DNA with a structure of A-linker-B, wherein A is a reverse-complement single-stranded DNA of the single-stranded DNA of SEQ ID NO: 11 at positions 68-90, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a single-stranded DNA of the sequence at positions 32-51 of SEQ ID NO: 11;
  • MTC primer BIP-Z comprises either of:
  • A-linker-B a single-stranded DNA with a structure of A-linker-B, wherein A is a single-stranded DNA of SEQ ID NO: 11 at positions 113-130, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a reverse-complement single-stranded DNA of the single-stranded DNA at positions 147-167 of SEQ ID NO: 11;
  • MTC primer LF-Z comprises either of:
  • MTC primer LB-Z comprises either of:
  • the MTC primer F3-F comprises or is the antisense DNA of the MTC primer F3-Z
  • the MTC primer B3-F comprises or is the antisense DNA of the MTC primer B3-Z
  • the MTC primer FIP-F comprises or is the antisense DNA of the MTC primer FIP-Z
  • the MTC primer BIP-F comprises or is the antisense DNA of the MTC primer BIP-Z
  • the MTC primer LF-F comprises or is the antisense DNA of the MTC primer LF-Z
  • the MTC primer LB-F comprises or is the antisense DNA of the MTC primer LB-Z
  • the primer set for detecting MP comprises six sense primers: MP primer F3-Z, MP primer B3-Z, MP primer FIP-Z, MP primer BIP-Z, MP primer LF-Z, and MP primer LB-Z; six antisense primers: MP primer F3-F, MP primer B3-F, MP primer FIP-F, MP primer BIP-F, MP primer LF-F, and MP primer LB-F; four sense primers: MP primer F3-Z, MP primer B3-Z, MP primer FIP-Z, and MP primer BIP-Z; or four antisense primers: MP primer F3-F, MP primer B3-F, MP primer FIP-F, and MP primer BIP-F, wherein the MP primer F3-Z comprises either of:
  • m1 a single-stranded DNA of nucleotide of SEQ ID NO: 12 at positions 14-30;
  • MP primer B3-Z comprises either of:
  • MP primer FIP-Z comprises either of:
  • A-linker-B a single-stranded DNA with a structure of A-linker-B, wherein A is a reverse-complement single-stranded DNA of the single-stranded DNA of SEQ ID NO: 12 at positions 71-89, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a single-stranded DNA of the sequence at positions 31-51 of SEQ ID NO: 12;
  • MP primer BIP-Z comprises either of:
  • A-linker-B a single-stranded DNA with a structure of A-linker-B, wherein A is a single-stranded DNA of SEQ ID NO: 12 at positions 99-117, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a reverse-complement single-stranded DNA of the single-stranded DNA at positions 155-171 of SEQ ID NO: 12;
  • MP primer LF-Z comprises either of:
  • MP primer LB-Z comprises either of:
  • the MP primer F3-F comprises or is the antisense DNA of the MP primer F3-Z
  • the MP primer B3-F comprises or is the antisense DNA of the MP primer B3-Z
  • the MP primer FIP-F comprises or is the antisense DNA of the MP primer FIP-Z
  • the MP primer BIP-F comprises or is the antisense DNA of the MP primer BIP-Z
  • the MP primer LF-F comprises or is the antisense DNA of the MP primer LF-Z
  • the MP primer LB-F comprises or is the antisense DNA of the MP primer LB-Z
  • the primer set for detecting CP comprises six sense primers: CP primer F3-Z, CP primer B3-Z, CP primer FIP-Z, CP primer BIP-Z, CP primer LF-Z, and CP primer LB-Z; six antisense primers: CP primer F3-F, CP primer B3-F, CP primer FIP-F, CP primer BIP-F, CP primer LF-F, and CP primer LB-F; four sense primers: CP primer F3-Z, CP primer B3-Z, CP primer FIP-Z, and CP primer BIP-Z; or four antisense primers: CP primer F3-F, CP primer B3-F, CP primer FIP-F, and CP primer BIP-F, wherein the CP primer F3-Z comprises either of:
  • n1 a single-stranded DNA of nucleotide of SEQ ID NO: 13 at positions 7-30;
  • n2) a single-stranded DNA with over 85%of identity with the single-stranded DNA of n1) ,
  • CP primer B3-Z comprises either of:
  • n5 a single-stranded DNA with over 85%of identity with the single-stranded DNA of n4)
  • CP primer FIP-Z comprises either of:
  • A-linker-B a single-stranded DNA with a structure of A-linker-B, wherein A is a reverse-complement single-stranded DNA of the single-stranded DNA of SEQ ID NO: 13 at positions 76-95, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a single-stranded DNA of the sequence at positions 35-54 of SEQ ID NO: 13;
  • CP primer BIP-Z comprises either of:
  • n10) a single-stranded DNA with a structure of A-linker-B, wherein A is a single-stranded DNA of SEQ ID NO: 13 at positions 127-152, the linker is a single-stranded DNA with a length of 0-10 nucleotides, and B is a reverse-complement single-stranded DNA of the single-stranded DNA at positions 183-202 of SEQ ID NO: 13;
  • n11 a single-stranded DNA with over 85%of identity with the single-stranded DNA of n10) ,
  • CP primer LF-Z comprises either of:
  • n14 a single-stranded DNA with over 85%of identity with the single-stranded DNA of n13) ,
  • CP primer LB-Z comprises either of:
  • n17 a single-stranded DNA fragment with over 85%of identity with the single-stranded DNA of n16) ,
  • the CP primer F3-F comprises or is the antisense DNA of the CP primer F3-Z
  • the CP primer B3-F comprises or is the antisense DNA of the CP primer B3-Z
  • the CP primer FIP-F comprises or is the antisense DNA of the CP primer FIP-Z
  • the CP primer BIP-F comprises or is the antisense DNA of the CP primer BIP-Z
  • the CP primer LF-F comprises or is the antisense DNA of the CP primer LF-Z
  • the CP primer LB-F comprises or is the antisense DNA of the CP primer LB-Z.
  • Embodiment 2 The primer set of Embodiment 1, wherein the nucleotide sequence of the linker in the SP primer FIP-Z is GTAG, the nucleotide sequence of the linker in the SA primer FIP-Z is AACAAC, the nucleotide length of the linker in SP primer FIP-Z is 0, the nucleotide length of the linker in the SP primer BIP-Z is 0, the nucleotide length of the linker in the MRSA primer FIP-Z is 0, the nucleotide length of the linker in the MRSA primer BIP-Z is 0, the nucleotide sequence of the linker in the E. coli primer FIP-Z is CT, the nucleotide sequence of the linker in the E.
  • coli primer BIP-Z is TTT
  • the nucleotide length of the linker in the KP primer FIP-Z is 0, the nucleotide sequence of the linker in the KP primer BIP-Z is TTTT
  • the nucleotide length of the linker in the PA primer FIP-Z is 0, the nucleotide length of the linker in the PA primer BIP-Z is 0, the nucleotide length of the AB primer FIP-Z is 0, the nucleotide length of the linker in the AB primer BIP-Z is 0, the nucleotide length of the linker in the AB primer BIP-Z is 0, the nucleotide length of the linker in the SMA primer FIP-Z is 0, the nucleotide length of the linker in the SMA primer BIP-Z is 0, the nucleotide sequence of the linker in the HI primer FIP-Z is ATCAAC
  • the nucleotide length of the linker in HI primer BIP-Z is
  • Embodiment 3 The primer set of Embodiment 1 or 2, wherein the molar ratio of the primers in each sense primer sets of six primers is: “F3-Z” 0.2-0.3 ⁇ mol (e.g., 0.2 ⁇ mol) , “B3-Z” 0.2-0.3 ⁇ mol, “FIP-Z” 0.8-2.4 ⁇ mol, “BIP-Z” 0.8-2.4 ⁇ mol, “LF-Z” 0.4-1.0 ⁇ mol, and/or “LB-Z” 0.4-1.0 ⁇ mol; the molar ratio of the primers in each sense primer sets of four primers is: “F3-Z” 0.2-0.3 ⁇ mol (e.g., 0.2 ⁇ mol) , “B3-Z” 0.2-0.3 ⁇ mol, “FIP-Z” 0.8-2.4 ⁇ mol, and/or “BIP-Z” 0.8-2.4 ⁇ mol; the molar ratio of the primers in each antisense primer sets of six primers is: “F3-F
  • Embodiment 4 A chip for detecting respiratory tract infection pathogens, comprising the LAMP primer for detecting respiratory tract infection pathogens of any of Embodiments 1-3, wherein the respiratory tract infection (RTI) -related pathogenic microorganisms are the following thirteen sets, any twelve sets, any eleven sets, any ten sets, any nine sets, any eight sets, any seven sets, any six sets, any five sets, any four sets, any three sets, any two sets or any one set: Streptococcus pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Haemophilus influenzae, Legionella pneumophila, Mycobacterium tuberculosis complex, mycoplasma pneumoniae, and Chlamydia pneumonia.
  • RTI respiratory tract
  • Embodiment 5 The fluidic chip of Embodiment 4, wherein the chip is an isothermal amplification micro-fluidic chip.
  • Embodiment 6 A system for detecting respiratory tract infection pathogens, comprising the LAMP primer for detecting respiratory tract infection pathogens of any of Embodiments 1-3, wherein the respiratory tract infection (RTI) -related pathogenic microorganisms are the following thirteen sets, any twelve sets, any eleven sets, any ten sets, any nine sets, any eight sets, any seven sets, any six sets, any five sets, any four sets, any three sets, any two sets, or any one set: Streptococcus pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Haemophilus influenzae, Legionella pneumophila, Mycobacterium tuberculosis complex, mycoplasma pneumoniae, and Chlamydia pneumonia.
  • RTI respiratory
  • Embodiment 7 The system of Embodiment 6, wherein the system comprises the fluidic chip for detecting respiratory tract infection pathogens of Embodiment 3 or 4.
  • Embodiment 8 The system of Embodiment 6 or 7, wherein the system comprises a reagent for detecting the respiratory tract infection pathogen (s) , and/or an instrument, and/or an amplification product data processor, wherein the reagent does not comprise a LAMP primer of any of Embodiments 1-3, the amplified product data processor is used to distinguish whether the test sample of LAMP amplification contains the specific amplification products by the LAMP primers of Embodiment 1 or 2.
  • the system comprises a reagent for detecting the respiratory tract infection pathogen (s) , and/or an instrument, and/or an amplification product data processor, wherein the reagent does not comprise a LAMP primer of any of Embodiments 1-3, the amplified product data processor is used to distinguish whether the test sample of LAMP amplification contains the specific amplification products by the LAMP primers of Embodiment 1 or 2.
  • Embodiment 9 A method of manufacturing the primer sets, the chip, and/or the system of a) -c) , respectively, comprising preparing at least one or all of the primers of any of Embodiments 1-3 for detecting the respiratory tract infection pathogen:
  • Embodiment 10 The applications of LAMP primers to detect the respiratory tract infection pathogenic microorganisms of any of Embodiments 1-3, comprising:
  • Embodiments 1-3 using the LAMP primers of any of Embodiments 1-3 to detect the respiratory tract infection pathogenic microorganisms used in the preparation of a reagent or kit to detect the respiratory tract infection pathogenic microorganisms,
  • RTI respiratory tract infection
  • the respiratory tract infection (RTI) -related pathogenic microorganisms are the following thirteen sets, any twelve sets, any eleven sets, any ten sets, any nine sets, any eight sets, any seven sets, any six sets, any five sets, any four sets, any three sets, any two sets or any one set: Streptococcus pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Haemophilus influenzae, Legionella pneumophila, Mycobacterium tuberculosis complex, mycoplasma pneumoniae, and Chlamydia pneumonia.

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Abstract

L'invention concerne des amorces LAMP destinées à détecter le(s) pathogène(s) dans une infection des voies respiratoires, et des kits, des puces et des systèmes comprenant lesdites amorces LAMP. Un procédé de détection d'un pathogène (p. ex., un pathogène dans une infection des voies respiratoires) ou de confirmation de l'identité du pathogène, au moyen d'amorces LAMP immobilisées sur une biopuce, est en outre décrit.
PCT/CN2016/000245 2015-05-08 2016-05-06 Compositions et procédés de détection de pathogènes dans les infections des voies respiratoires WO2016180037A1 (fr)

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