CN110656188A - Primer and/or probe composition for detecting bacillus causing bloodstream infection and application thereof - Google Patents
Primer and/or probe composition for detecting bacillus causing bloodstream infection and application thereof Download PDFInfo
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Abstract
Primer and/or probe compositions for detecting bacilli that cause bloodstream infections and uses thereof are provided. The application particularly relates to a primer and/or probe composition or related reagent or kit for detecting one or more of mycobacterium tuberculosis, listeria monocytogenes, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, escherichia coli, haemophilus influenzae and proteus mirabilis, so that the detection period of the bacilli is greatly shortened, the efficiency and the detection flux are improved, and the detection requirements are met. In addition, the method and the related products thereof have higher specificity, sensitivity and accuracy.
Description
Technical Field
The present application belongs to the field of molecular biology and in particular relates to primer and/or probe compositions for the detection of bacilli, in particular gram-negative bacilli, that cause infections in the bloodstream, and uses thereof.
Background
Bloodstream infection (BSI) refers to a systemic inflammatory response, infection and poisoning caused by various pathogenic bacteria entering the blood circulation, and is a serious life-threatening infectious disease. Because the disease has a hidden onset and a rapid development, if the disease is not treated effectively in time, the life of the patient is threatened. However, the clinical manifestations of bloodstream infection lack specificity, and are prone to missed diagnosis and misdiagnosis, so early diagnosis of bloodstream infection is of great clinical significance.
Pathogenic bacteria causing bloodstream infections are mainly classified into three groups, gram-positive bacteria, gram-negative bacteria and fungi. Among the common pathogenic bacteria of bloodstream infection, the common bacilli are mainly gram-negative bacteria, including klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, escherichia coli, haemophilus influenzae, proteus mirabilis, etc., while listeria monocytogenes is gram-positive bacteria. In addition, M.tuberculosis is also a common causative bacterium of bloodstream infections, but cannot be classified by gram methods because it cannot be stained with crystal violet.
Although the blood culture method can provide a basis for clinical pathogenic diagnosis and is the current 'gold standard' for diagnosing bacterial blood stream infection, only 30-40% of blood stream infected persons can find pathogenic bacteria by a culture mode, the positive detection rate of blood culture is not high, the detection time is too long, and the positive alarm needs 1-5 days or even longer. However, for clinical treatment, if early diagnosis and rational antibacterial treatment are not performed within 24 hours of the early stage of bloodstream infection, the survival rate of patients will be greatly reduced. The lack of accurate diagnosis results makes it difficult for clinicians to select the optimal treatment regimen, and blinding antibiotics may lead to adverse consequences such as decreased survival of patients, emergence of bacterial resistance, and increased treatment costs.
Therefore, there is a need for methods and related products that enable early diagnosis of bloodstream infections.
Disclosure of Invention
In order to address the above-mentioned problems, in a first aspect, the present application provides a primer and/or probe composition for detecting a bacillus causing a bloodstream infection, comprising a primer and/or probe selected from the group consisting of for detecting one or more of the following targets: specific insertion sequences (IS6110 sequence) of mycobacterium tuberculosis complex, listeria monocytogenes hemolysin gene (hlyA gene), klebsiella pneumoniae outer membrane phosphoprotein gene (phoE gene), pseudomonas aeruginosa ETA protein coding gene (toxA gene), acinetobacter baumannii oxacillin gene (OXA51 gene), escherichia coli putative protein gene (ydiJ gene), haemophilus influenzae outer membrane protein P6 gene (ompP6 gene), and proteus mirabilis urease synthesis regulatory factor gene (ureR gene).
In particular embodiments, the primer and/or probe compositions provided herein for detecting a bacillus that primes a bloodstream infection comprise any two, three, four, five, six, seven, or eight of the primers and/or probes for detecting the IS6110 sequence, the hlyA gene, the phoE gene, the toxA gene, the OXA51 gene, the ydiJ gene, the ompP6 gene, and the ureR gene, respectively.
For example, in one embodiment, the primer and/or probe compositions provided herein for detecting a bacterium that causes a bloodstream infection comprise a primer and/or probe for detecting the IS6110 sequence and/or the hlyA gene, respectively.
In one embodiment, the primer and/or probe compositions provided herein for detecting a bacillus that primes a bloodstream infection comprise primers and/or probes for detecting the phoE gene and/or the toxA gene, respectively.
In one embodiment, the primer and/or probe compositions provided herein for detecting a bacillus that primes a bloodstream infection comprise primers and/or probes for detecting the OXA51 gene and/or the ydiJ gene, respectively.
In one embodiment, the primer and/or probe composition for detecting a bacillus causing a bloodstream infection provided herein comprises a primer and/or probe for detecting the ompP6 gene and/or the ureR gene, respectively.
In another embodiment, in the primer and/or probe composition for detecting bacilli causing bloodstream infection provided by the present application, the primer sequence for detecting said IS6110 sequence IS shown as SEQ ID NO.1 and SEQ ID NO.2, and the probe sequence IS shown as SEQ ID NO. 3.
In another embodiment, the primer and/or probe composition for detecting bacillus causing bloodstream infection, provided by the application, has the primer sequence shown in SEQ ID NO.4 and SEQ ID NO.5 and the probe sequence shown in SEQ ID NO.6 for detecting the hlyA gene;
in another embodiment, the primer and/or probe composition for detecting a bacillus which initiates bloodstream infection, provided herein, has the primer sequences shown in SEQ ID No.7 and SEQ ID No.8 and the probe sequence shown in SEQ ID No.9 for detecting the phoE gene;
in another embodiment, in the primer and/or probe composition for detecting bacillus causing bloodstream infection provided by the present application, the primer sequence for detecting the toxA gene is shown as SEQ ID No.10 and SEQ ID No.11, and the probe sequence is shown as SEQ ID No. 12;
in another embodiment, in the primer and/or probe composition for detecting bacillus which initiates bloodstream infection provided by the present application, the primer sequence for detecting said OXA51 gene is shown as SEQ ID No.13 and SEQ ID No.14, and the probe sequence is shown as SEQ ID No. 15;
in another embodiment, in the primer and/or probe composition for detecting bacilli that cause bloodstream infection provided herein, the primer sequences for detecting the ydiJ gene are shown as SEQ ID No.16 and SEQ ID No.17, and the probe sequence is shown as SEQ ID No. 18;
in another embodiment, the primer and/or probe composition for detecting bacteria that cause bloodstream infection, provided herein, has the primer sequence shown in SEQ ID NO.19 and SEQ ID NO.20 and the probe sequence shown in SEQ ID NO.21 for detecting the ompP6 gene.
In another embodiment, the primer and/or probe composition for detecting bacilli that cause bloodstream infection provided herein, the primer sequences for detecting the ureR gene are shown as SEQ ID No.22 and SEQ ID No.23, and the probe sequence is shown as SEQ ID No. 24.
In yet another embodiment, provided herein are primer and/or probe compositions for detecting a bacillus that causes a bloodstream infection, wherein the probe sequences are labeled with a fluorescent reporter group and a quencher group, respectively. In particular embodiments, the fluorescent reporter group is selected from FAM, ROX, JOE, HEX or TET and the quenching group is selected from BHQ1, BHQ2 or TAMRA.
In yet another embodiment, the primer and/or probe compositions provided herein for detecting a bacillus that primes a bloodstream infection further comprise deoxyribonucleoside triphosphates dn (u) TPs.
In a second aspect, the present application provides the use of a primer and/or probe composition according to any one of the embodiments above in the preparation of a reagent or kit for the detection of a bacterium that causes an infection in the bloodstream.
In a third aspect, the present application provides a reagent or kit for detecting a bacillus causing a bloodstream infection, comprising a primer and/or probe composition according to any one of the embodiments above.
In one embodiment, the reagents or kits provided herein further comprise primers and/or probes for detecting an internal standard gene. In a specific embodiment, the internal reference gene is an int gene. In another specific embodiment, the primer sequence for detecting the int gene of the internal standard gene is shown as SEQ ID NO.25 and SEQ ID NO.26, and the probe sequence is shown as SEQ ID NO. 27.
In another embodiment, the reagents or kits provided herein further comprise one or more of: nucleic acid extract, PCR reaction enzyme system, negative control or positive control. In particular embodiments, the nucleic acid extract comprises: 2% (M/V) Chelex-100 and 1% (V/V) Tris-HCl. In another embodiment, the PCR reaction enzyme system comprises: 5U/. mu.L Taq DNA polymerase and 2U/. mu.L uracil-N-glycosylase. In another embodiment, the negative control is purified double distilled water. In another specific embodiment, the positive control is a T-vector plasmid carrying the target to be detected or an engineered escherichia coli bacterium containing the T-vector plasmid carrying the target to be detected.
In a fourth aspect, the present application provides a method for detecting a bacillus causing a bloodstream infection, comprising detecting a biological sample using a primer and/or probe composition according to any one of the embodiments above or a reagent or kit according to any one of the embodiments above.
In a fifth aspect, the present application provides primers and/or probes for the detection of mycobacterium tuberculosis. In one embodiment, the target of the primers and/or probes for detection of M.tuberculosis IS an insertion sequence (IS6110 sequence) specific for M.tuberculosis complex. In one embodiment, the primer sequences for detecting the IS6110 sequence are shown as SEQ ID NO.1 and SEQ ID NO. 2. In another embodiment, the probe sequence for detecting the IS6110 sequence IS shown in SEQ ID NO. 3. In yet another embodiment, the probe sequence for detecting the IS6110 sequence IS labeled with a fluorescent reporter FAM at the 5 'end and a quencher BHQ1 at the 3' end.
Further, the present application provides a reagent or a kit for detecting Mycobacterium tuberculosis, which comprises a primer and/or a probe for detecting an insertion sequence (IS6110 sequence) specific to Mycobacterium tuberculosis complex in any of the above embodiments.
In a sixth aspect, the present application provides primers and/or probes for detecting listeria monocytogenes. In one embodiment, the target of the primers and/or probes for detecting listeria monocytogenes is the listeria monocytogenes hemolysin gene (hlyA gene). In one embodiment, the primer sequences for detecting the hlyA gene are shown as SEQ ID No.4 and SEQ ID No. 5. In another embodiment, the probe sequence for detecting the hlyA gene is shown as SEQ ID NO. 6. In yet another embodiment, the probe sequence for detecting the hlyA gene is labeled with a fluorescent reporter ROX at the 5 'end and a quencher BHQ2 at the 3' end.
Further, the present application provides a reagent or a kit for detecting listeria monocytogenes, which comprises a primer and/or a probe for detecting listeria monocytogenes hemolysin gene (hlyA gene) in any of the above embodiments.
In a seventh aspect, the present application provides primers and/or probes for the detection of klebsiella pneumoniae. In one embodiment, the target of the primers and/or probes used for the detection of klebsiella pneumoniae is the klebsiella pneumoniae outer membrane phosphoprotein gene (phoE gene). In one embodiment, the primer sequences for detecting the phoE gene are shown in SEQ ID NO.7 and SEQ ID NO. 8. In another embodiment, the probe sequence for detecting the phoE gene is shown in SEQ ID NO. 9. In yet another embodiment, the probe sequence for detecting the phoE gene is labeled with a fluorescent reporter FAM at the 5 'end and a quencher BHQ1 at the 3' end.
Further, the present application provides a reagent or a kit for detecting klebsiella pneumoniae, which comprises a primer and/or a probe for detecting klebsiella pneumoniae outer membrane phosphoprotein gene (phoE gene) in any one of the above embodiments.
In an eighth aspect, the present application provides primers and/or probes for use in the detection of pseudomonas aeruginosa. In one embodiment, the primer and/or probe used to detect pseudomonas aeruginosa is targeted to the pseudomonas aeruginosa ETA protein encoding gene (toxA gene). In one embodiment, the primer sequences for detecting the toxA gene are shown as SEQ ID No.10 and SEQ ID No. 11. In another embodiment, the probe sequence for detecting the toxA gene is shown in SEQ ID No. 12. In yet another embodiment, the probe sequence for detecting the toxA gene is labeled with a fluorescent reporter ROX at the 5 'end and a quencher BHQ2 at the 3' end.
Further, the present application provides a reagent or kit for the detection of pseudomonas aeruginosa comprising primers and/or probes for the detection of the pseudomonas aeruginosa ETA protein encoding gene (toxA gene) in any of the embodiments described above.
In a ninth aspect, the present application provides primers and/or probes for the detection of acinetobacter baumannii. In one embodiment, the target of the primers and/or probes used to detect the immobile rod of baumannii is the acinetobacter baumannii oxacillin gene (OXA51 gene). In one embodiment, the primer sequences for detecting the OXA51 gene are shown as SEQ ID No.13 and SEQ ID No. 14. In another embodiment, the probe sequence for detecting the OXA51 gene is shown in SEQ ID No. 15. In yet another embodiment, the probe sequence for detecting the OXA51 gene is labeled with a fluorescent reporter group FAM at the 5 'end and a quencher group BHQ1 at the 3' end.
Further, the present application provides a reagent or a kit for detecting acinetobacter baumannii, which comprises a primer and/or a probe for detecting acinetobacter baumannii oxacillin enzyme gene (OXA51 gene) in any of the above embodiments.
In a tenth aspect, the present application provides primers and/or probes for the detection of escherichia coli. In one embodiment, the target of the primers and/or probes used for detection of escherichia coli is the escherichia coli putative protein gene (ydiJ gene). In one embodiment, the primer sequences for detecting the ydiJ gene are shown in SEQ ID NO.16 and SEQ ID NO. 17. In another embodiment, the probe sequence for detecting the ydiJ gene is shown in SEQ ID NO. 18. In yet another embodiment, the probe sequence for detecting the ydiJ gene is labeled at the 5 'end with a fluorescent reporter ROX and at the 3' end with a quencher BHQ 2.
Further, the present application provides a reagent or a kit for detecting escherichia coli, which comprises a primer and/or a probe for detecting a putative protein gene (ydiJ gene) of escherichia coli in any of the above embodiments.
In an eleventh aspect, the present application provides primers and/or probes for detecting the haemophilus influenzae outer membrane protein P6 gene (ompP6 gene). In one embodiment, the target of the primers and/or probes for detecting haemophilus influenzae is the haemophilus influenzae outer membrane protein P6 gene (ompP6 gene). In one embodiment, the primer sequences for detecting the ompP6 gene are shown in SEQ ID NO.19 and SEQ ID NO. 20. In another embodiment, the probe sequence for detecting the ompP6 gene is shown in SEQ ID NO. 21. In yet another embodiment, the probe sequence for detecting the ompP6 gene is labeled with a fluorescent reporter FAM at the 5 'end and a quencher BHQ1 at the 3' end.
Further, the present application provides a reagent or a kit for detecting Haemophilus influenzae, which comprises a primer and/or a probe for detecting outer membrane protein P6 gene (ompP6 gene) of Haemophilus influenzae in any of the above embodiments.
In a twelfth aspect, the present application provides primers and/or probes for detecting proteus mirabilis. In one embodiment, the target of the primers and/or probes used to detect proteus mirabilis is the proteus mirabilis urease synthesis regulator gene (ureR gene). In one embodiment, the primer sequences for detecting the ureR gene are shown in SEQ ID NO.22 and SEQ ID NO. 23. In another embodiment, the probe sequence for detecting the ureR gene is set forth in SEQ ID NO. 24. In yet another embodiment, the probe sequence for detecting the ureR gene is labeled with a fluorescent reporter group ROX at the 5 'end and a quencher group BHQ2 at the 3' end.
Further, the present application provides a reagent or a kit for detecting proteus mirabilis, which comprises a primer and/or a probe for detecting a proteus mirabilis urease synthesis regulatory factor gene (ureR gene) in any of the above embodiments.
In a thirteenth aspect, the present application provides primer and/or probe compositions for the detection of mycobacterium tuberculosis and listeria monocytogenes. In one embodiment, the target of the primer and/or probe for detecting mycobacterium tuberculosis IS a specific insertion sequence (IS6110 sequence) of mycobacterium tuberculosis complex, and the target of the primer and/or probe for detecting listeria monocytogenes IS a listeriolysin gene (hlyA gene). In a specific embodiment, the primer and/or probe composition comprises SEQ ID No.1 to 6. In another specific embodiment, the primer and/or probe composition further comprises a primer and/or probe for detecting an internal standard gene int gene. In yet another specific embodiment, said primer and/or probe composition further comprises SEQ ID No.25 to 27. In yet another specific embodiment, the primer and/or probe composition further comprises deoxyribonucleoside triphosphates dn (u) TP. In another specific embodiment, the primer and/or probe composition comprises the following components: 2 μ L10 XPCR buffer, 2 μ L25 mmol/L MgCl21.6. mu.L of 2.5mmol/L dN (U) TP, 0.6. mu.L of 10. mu. mol/L of SEQ ID NO.1 and 2 and SEQ ID NO.4 and 5, 0.2. mu.L of 10. mu. mol/L of SEQ ID NO.3 and 6, 0.6. mu.L of 10. mu. mol/L of SEQ ID NO.25 and 26, 0.2. mu.L of 10. mu. mol/L of SEQ ID NO.27, and 7. mu.L of sterilized purified water.
Further, the present application provides a reagent or a kit for detecting mycobacterium tuberculosis and listeria monocytogenes, comprising a primer and/or a probe composition for detecting a unique insertion sequence (IS6110 sequence) of mycobacterium tuberculosis complex and a listeria monocytogenes hemolysin gene (hlyA gene) in any of the above embodiments.
In a fourteenth aspect, the present application provides methods for detectingPrimer and/or probe compositions for klebsiella pneumoniae and pseudomonas aeruginosa ETA protein coding genes (toxA genes). In one embodiment, the primer and/or probe for detecting klebsiella pneumoniae targets the klebsiella pneumoniae outer membrane phosphoprotein gene (phoE gene), and the primer and/or probe for detecting pseudomonas aeruginosa targets the pseudomonas aeruginosa ETA protein coding gene (toxA gene). In a specific embodiment, the primer and/or probe composition comprises SEQ ID No.7 to 12. In another specific embodiment, the primer and/or probe composition further comprises a primer and/or probe for detecting an internal standard gene int gene. In yet another specific embodiment, said primer and/or probe composition further comprises SEQ ID No.25 to 27. In yet another specific embodiment, the primer and/or probe composition further comprises deoxyribonucleoside triphosphates dn (u) TP. In another specific embodiment, the primer and/or probe composition comprises the following components: 2 μ L10 XPCR buffer, 2 μ L25 mmol/L MgCl21.6. mu.L of 2.5mmol/L dN (U) TP, 0.6. mu.L of 10. mu. mol/L of SEQ ID NO.7 and 8 and SEQ ID NO.10 and 11, 0.2. mu.L of 10. mu. mol/L of SEQ ID NO.9 and 12, 0.6. mu.L of 10. mu. mol/L of SEQ ID NO.25 and 26, 0.2. mu.L of 10. mu. mol/L of SEQ ID NO.27, and 7. mu.L of sterilized purified water.
Further, the present application provides a reagent or kit for detecting klebsiella pneumoniae and pseudomonas aeruginosa, comprising a primer and/or probe composition for detecting klebsiella pneumoniae outer membrane phosphoprotein gene (phoE gene) and pseudomonas aeruginosa ETA protein coding gene (toxA gene) in any of the above embodiments.
In a fifteenth aspect, the present application provides primer and/or probe compositions for detecting the acinetobacter baumannii oxacillin gene (OXA51 gene) and the escherichia coli putative protein gene (ydiJ gene). In one embodiment, the target of the primers and/or probes for detecting acinetobacter baumannii is acinetobacter baumannii oxacillin gene (OXA51 gene), and the target of the primers and/or probes for detecting escherichia coli is escherichia coli putative protein gene (ydiJ gene). In a specific embodiment, the primer and/or probe composition comprises SEQID Nos. 13 to 18. In another specific embodiment, the primer and/or probe composition further comprises a primer and/or probe for detecting an internal standard gene int gene. In yet another specific embodiment, said primer and/or probe composition further comprises SEQ ID No.25 to 27. In yet another specific embodiment, the primer and/or probe composition further comprises deoxyribonucleoside triphosphates dn (u) TP. In another specific embodiment, the primer and/or probe composition comprises the following components: 2 μ L10 XPCR buffer, 2 μ L25 mmol/L MgCl21.6. mu.L of 2.5mmol/L dN (U) TP, 0.6. mu.L of 10. mu. mol/L of SEQ ID NO.13 and 14 and SEQ ID NO.16 and 17, 0.2. mu.L of 10. mu. mol/L of SEQ ID NO.15 and 18, 0.6. mu.L of 10. mu. mol/L of SEQ ID NO.25 and 26, 0.2. mu.L of 10. mu. mol/L of SEQ ID NO.27, and 7. mu.L of sterilized purified water.
Further, the present application provides a reagent or a kit for detecting acinetobacter baumannii and escherichia coli, which comprises a primer and/or probe composition for detecting acinetobacter baumannii oxacillin enzyme gene (OXA51 gene) and escherichia coli putative protein gene (ydiJ gene) in any of the above embodiments.
In a sixteenth aspect, the present application provides primer and/or probe compositions for haemophilus influenzae and proteus mirabilis. In one embodiment, the primer and/or probe for detecting haemophilus influenzae is targeted to the haemophilus influenzae outer membrane protein P6 gene (ompP6 gene) and the primer and/or probe for detecting proteus mirabilis is targeted to the proteus mirabilis urease synthesis regulator gene (ureR gene). In a specific embodiment, the primer and/or probe composition comprises SEQ ID nos. 19 to 24. In another specific embodiment, the primer and/or probe composition further comprises a primer and/or probe for detecting an internal standard gene int gene. In yet another specific embodiment, said primer and/or probe composition further comprises SEQ ID No.25 to 27. In yet another specific embodiment, the primer and/or probe composition further comprises deoxyribonucleoside triphosphates dn (u) TP. In another specific embodiment, the primer and/or probe composition comprises the following components: 2 μ L10 XPCR buffer, 2 μ L25 mmol/L MgCl21.6. mu.L 2.5mmol/L dN (U) TP, 0.6. mu.L 10. mu. mol/L of SEQ ID NO.19 and 20 and SEQ ID NO.22 and 23, 0.2. mu.L 10. mu. mol/L of SEQ ID NO.21 and 24, 0.6. mu.L 10. mu. mol/L of SEQ ID NO.25 and 26, 0.2. mu.L 10. mu. mol/L of SEQ ID NO.27, and 7. mu.L sterilized purified water.
Further, the present application provides a reagent or a kit for detecting haemophilus influenzae and proteus mirabilis, which comprises a primer and/or probe composition for detecting outer membrane protein P6 gene (ompP6 gene) of haemophilus influenzae and urease synthesis regulator gene (ureR gene) of proteus mirabilis in any of the above embodiments.
In a seventeenth aspect, the present application provides a method of using the above reagent or kit.
It can be seen that the present application provides primer and/or probe compositions and related detection reagents or kits for detecting bacilli that cause blood stream infections, including any one or more of mycobacterium tuberculosis, listeria monocytogenes, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, escherichia coli, haemophilus influenzae, and proteus mirabilis, using real-time fluorescent quantitative PCR to detect biological samples.
Compared with the conventional clinical detection method, the method provided by the application has the advantages that the detection period is greatly shortened, the efficiency and the detection flux are improved, and the detection requirement is met. In addition, the primer and/or probe composition and the related detection reagent or kit provided by the application have high specificity, sensitivity and accuracy, and meanwhile, the pollution problem in the experiment can be effectively solved because the detection process after amplification is not carried out in the experiment.
Drawings
FIG. 1 IS a fluorescent quantitative PCR graph showing the amplification results of the positive control of IS6110 sequence and hlyA gene in example 2
FIG. 2 is a fluorescent quantitative PCR graph showing the results of positive control amplification of the phoE gene and the toxA gene in example 2
FIG. 3 is a fluorescent quantitative PCR plot of the positive control amplification results of the OXA51 gene and the ydiJ gene in example 2;
FIG. 4 is a fluorescent quantitative PCR graph showing the amplification results of the positive controls of ompP6 gene and ureR gene in example 2.
Detailed description of the preferred embodiments
In order to realize early diagnosis of blood stream infection, the inventor carries out deep research on various main bacilli causing blood stream infection, including mycobacterium tuberculosis, listeria monocytogenes, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, escherichia coli, haemophilus influenzae and proteus mirabilis, and finally respectively screens and determines the following detection target genes: the mycobacterium tuberculosis detection target IS a specific insertion sequence (IS6110 sequence) of a mycobacterium tuberculosis complex, the listeria monocytogenes detection target IS a hemolysin gene (hlyA gene), the klebsiella pneumoniae detection target IS an outer membrane phosphoprotein gene (phoE gene), the pseudomonas aeruginosa detection target IS an ETA protein coding gene (toxA gene), the acinetobacter baumannii detection target IS a oxacillin gene (OXA51 gene), the escherichia coli detection target IS a hypothetical protein gene (ydiJ gene), the haemophilus influenzae detection target IS an outer membrane protein P6 gene (ompP6 gene), and the proteus mirabilis detection target IS a urease synthesis regulatory factor gene (ureR gene).
On the basis, the inventor further selects conservative and specific segments of each gene through sequence comparison, designs a plurality of specific primers and probes for detecting target genes respectively, and screens out sequences capable of effectively avoiding mutual interference between the primers during co-tube amplification, thereby establishing a multiple fluorescence quantitative PCR reaction system with good specificity and sensitivity.
Specifically, the sequences of the detection primers and the probes determined by screening are as follows:
1) according to the IS6110 insertion sequence (Genebank accession number: y14048.1) and one probe as follows:
| forward primer (SEQ ID NO.1) | 5’-GTCTACTTGGTGTTGGCT-3’ |
| Reverse primer (SEQ ID NO.2) | 5’-CTGATGATCGGCGATGAA-3’ |
| Probe (SEQ ID NO.3) | 5’-FAM-ATTGCGAAGGGCGAACGCGATT-BHQ1-3’ |
2) According to the hlyA gene sequence (Genebank accession number: GU810924.1) were designed as follows:
| forward primer (SEQ ID NO.4) | 5’-AACTTCAAAAGCTTATACAGATGGAAA-3’ |
| Reverse primer (SEQ ID NO.5) | 5’-GGCAAATAGATGGACGATGTGAA-3’ |
| Probe (SEQ ID NO.6) | 5’-ROX-ATCACTCTGGAGGATACGTTGCTCAAT-BHQ2-3’ |
3) According to the phoE gene sequence (Genebank accession number: AF009172.1) was designed as follows:
| forward primer (SEQ ID NO.7) | 5’-CCTGAAATATGACGCCAACAAT-3’ |
| Reverse primer (SEQ ID NO.8) | 5’-ACCGAAGTCGAACTGATACTG-3’ |
| Probe (SEQ ID NO.9) | 5’-FAM-CGACCATGTACTCTGAAACCCGCA-BHQ1-3’ |
4) According to the toxA gene sequence (Genebank accession number: KC847704.1) and one probe as follows:
| forward primer (SEQ ID NO.10) | 5’-AGAAGCCTTCGAACATCAAGGT-3’ |
| Reverse primer (SEQ ID NO.11) | 5’-GGTGTAGATCGGCGACATGTG-3’ |
| Probe (SEQ ID NO.12) | 5’-ROX-TCATCCACGAACTGAACGCCGGTAAC-BHQ2-3’ |
5) According to the OXA51 gene sequence (Genebank accession number: NG — 049677.1) were designed as follows:
| forward primer (SEQ ID NO.13) | 5’-CCGGTTTATCAAGATTTAGC-3’ |
| Reverse primer (SEQ ID NO.14) | 5’-GTACCCAAGTCGATAATTTTTG-3’ |
| Probe (SEQ ID NO.15) | 5’-FAM-GTCTAATGAAGTGAAGCGTGTTGGTT-BHQ1-3’ |
6) According to the ydiJ gene sequence (Genebank accession number: AE005174.2) were designed as follows:
| forward primer (SEQ ID NO.16) | 5’-CGGTGAATGAATCCGGTTCT-3’ |
| Reverse primer (SEQ ID NO.17) | 5’-TGTAACGGCAACGGTTTATGC-3’ |
| Probe (SEQ ID NO.18) | 5’-ROX-CATCGACGGACACATCGGACTACGG-BHQ2-3’ |
7) According to the ompP6 gene sequence (Genebank accession number: HM124552.1) was designed as follows:
| forward primer (SEQ ID NO.19) | 5'-GGTACACCAGAATACAACATCG-3' |
| Reverse primer (SEQ ID NO.20) | 5'-TGACCTAATACTGCAGGTTTTTC-3' |
| Probe (SEQ ID NO.21) | 5'-FAM-CTAATTTACCAGCATCAACACCTTTACC-BHQ1-3' |
8) According to the ureR gene sequence (Genebank accession number: AM942759.1) was designed as follows:
| forward primer (SEQ ID NO.22) | 5’-AACCGATTGAATTACATACCTTAGTAC-3’ |
| Reverse primer (SEQ ID NO.23) | 5’-TGTTGCATAAACAGGGTCTCT-3’ |
| Probe (SEQ ID NO.24) | 5’-ROX-CCATGTTCAAGCGGTAGACATTGCTGAAGTAAC-BHQ2-3’ |
9) In addition, an internal standard gene (int) is further provided for identifying causes of undesirable results due to instrument failure, reagent factors, polymerase activity factors, presence of inhibitors in a sample, or the like. The inventor selects the ITS1 gene segment in rice ribosome DNA as an internal standard, compares the int gene with the detection target gene sequence, selects a specific segment and designs a specific primer and a fluorescent labeled probe aiming at the int gene as follows:
| forward primer (SEQ ID NO.25) | 5’-GCGATACCACGAGCTAAATC-3’ |
| Reverse primer (SEQ ID NO.26) | 5’-GCATTTCGCTACGTTCTTCAT-3’ |
| Probe (SEQ ID NO.27) | 5’-JOE-ACTCTCGGCAACGGATATCTCGGCTC-TAMRA-3’ |
On the basis, the application provides a kit for detecting common bacilli initiating bloodstream infection, which comprises nucleic acid extraction liquid, a PCR reaction enzyme system, an internal standard, a negative reference substance, a positive reference substance, a first primer probe mixed liquid, a second primer probe mixed liquid, a third primer probe mixed liquid and a fourth primer probe mixed liquid.
In one embodiment, the nucleic acid extract contained in the kit consists of 2% (M/V) Chelex-100, 1% (V/V) Tris-HCl, 1M, pH 9.0.
In one embodiment, the PCR reaction enzyme contained in the kit is composed of 5U/. mu.L Taq DNA polymerase and 2U/. mu.L uracil-N-glycosylase (UNG enzyme) mixed in a volume ratio of 3: 1.
In one embodiment, the internal standard comprised in the kit is a plasmid containing an internal standard int gene fragment.
In one embodiment, the negative control contained in the kit is double distilled water purified by a Millipore water purifier.
In one embodiment, the positive control contained in the kit IS a T-vector plasmid for detecting a target sequence of mycobacterium tuberculosis (IS6110), a T-vector plasmid for detecting a target gene of listeria monocytogenes (hlyA a), a T-vector plasmid for detecting a target gene of klebsiella pneumoniae (phoE), a T-vector plasmid for detecting a target gene of pseudomonas aeruginosa (toxA), a T-vector plasmid for detecting a target gene of acinetobacter baumannii (OXA51), a T-vector plasmid for detecting a target gene of escherichia coli (ydiJ), a T-vector plasmid for detecting a target gene of haemophilus influenzae (ompP6), a T-vector plasmid for detecting a target gene of proteus mirabilis (ureR), or an engineered escherichia coli containing the foregoing plasmids, and the bacterial fluid concentration as a control IS 105 CFU/mL.
In one embodiment, the first primer probe mixture contained in the kit comprises: the forward primer, reverse primer and probe for detecting said IS6110 sequence and said hlyA gene respectively constitute a first primer-probe composition. Further, the first primer-probe mixture further includes deoxyribonucleoside triphosphate dN (U) TP.
In a specific embodiment, the first primer probe mixture contained in the kit consists of 2. mu.L of 10 XPCR buffer, 2. mu.L of 25mmol/L MgCl2, 1.6. mu.L of 2.5mmol/L dN (U) TP, 0.6. mu.L of 10. mu. mol/L forward and reverse primers for detecting IS6110 sequence and hlyA gene, respectively, 0.2. mu.L of 10. mu. mol/L probe for detecting IS6110 sequence and hlyA gene, 0.6. mu.L of 10. mu. mol/L forward and reverse primers for detecting internal standard int gene, respectively, and 0.2. mu.L of 10. mu. mol/L probe for detecting internal standard int gene, respectively, and 7. mu.L sterilized purified water.
In one embodiment, the second primer probe mixture contained in the kit comprises: and the forward primer, the reverse primer and the probe which are respectively used for detecting the phoE gene and the toxA gene form a second primer probe composition. Further, the second primer probe mixture further comprises deoxyribonucleoside triphosphate dN (U) TP.
In a specific embodiment, the second primer probe mixture contained in the kit consists of 2. mu.L of 10 XPCR buffer, 2. mu.L of 25mmol/L MgCl2, 1.6. mu.L of 2.5mmol/L dN (U) TP, 0.6. mu.L of 10. mu. mol/L forward and reverse primers for detecting the phoE gene and the toxA gene, 0.2. mu.L of 10. mu. mol/L probe for detecting the phoE gene and the toxA gene, 0.6. mu.L of 10. mu. mol/L forward and reverse primers for detecting the internal standard int gene, 0.2. mu.L of 10. mu. mol/L probe for detecting the internal standard int gene, and 7. mu.L of sterilized purified water.
In one embodiment, the third primer probe mixture contained in the kit comprises: a forward primer, a reverse primer and a probe for detecting the OXA51 gene and the ydiJ gene, respectively. Further, the third primer-probe mixture further includes deoxyribonucleoside triphosphates dn (u) TP.
In a specific embodiment, the third primer probe mixture contained in the kit consists of 2 μ L of 10 × PCR buffer, 2 μ L of 25mmol/L MgCl2, 1.6 μ L of 2.5mmol/L dN (U) TP, 0.6 μ L of 10 μmol/L forward and reverse primers for detecting OXA51 gene and ydiJ gene, respectively, 0.2 μ L of 10 μmol/L probe for detecting OXA51 gene and ydiJ gene, 0.6 μ L of 10 μmol/L forward and reverse primers for detecting internal standard int gene, respectively, and 0.2 μ L of 10 μmol/L probe for detecting internal standard int gene, and 7 μ L of sterilized purified water.
In one embodiment, the fourth primer probe mixture comprises: a forward primer, a reverse primer and a probe for detecting the ompP6 gene and the ureR gene, respectively. Further, the fourth primer-probe mixture further includes deoxyribonucleoside triphosphates dn (u) TP.
In a specific embodiment, the fourth primer-probe mixture contained in the kit comprises 2 μ L of 10 XPCR buffer, 2 μ L of 25mmol/L MgCl2, 1.6 μ L of 2.5mmol/L dN (U) TP, 0.6 μ L of 10 μmol/L forward primer and reverse primer for detecting ompP6 gene and ureR gene respectively, 0.2 μ L of 10 μmol/L probe for detecting ompP6 gene and ureR gene respectively, 0.6 μ L of 10 μmol/L forward primer and reverse primer for detecting internal standard int gene respectively, and 0.2 μ L of 10 μmol/L probe for detecting internal standard int gene respectively, and 7 μ L of sterilized purified water.
In another embodiment, the kit comprises a package of reagent bottles or tubes that separate and collectively package the reagents.
In addition, the present application also provides a method of use of a kit for detecting common bacilli that cause bloodstream infections, comprising:
(1) preparing sample nucleic acid to obtain a nucleic acid membrane plate;
(2) placing the negative control and the positive control in a centrifuge tube, respectively adding the nucleic acid extraction solution, mixing, heating, centrifuging, and collecting the supernatant for fluorescence PCR detection;
(3) preparing an enzyme reagent, and mixing 5U/mu L of Taq DNA polymerase and 2U/mu L uracil-N-glycosylase (UNG enzyme) according to a volume ratio of 3: 1;
(4) preparing a PCR detection mixed solution: respectively shaking, uniformly mixing and centrifuging the prepared first, second, third or fourth primer probe mixed solution and an enzyme reagent;
(5) respectively placing the PCR detection mixed solution after adding the enzyme and mixing uniformly into a fluorescent PCR tube, sampling the nucleic acid extraction supernatant, the negative control nucleic acid extraction supernatant, the positive control nucleic acid extraction supernatant and the plasmid containing the int gene fragment of the internal standard into the fluorescent PCR tube of the existing PCR detection mixed solution, and carrying out fluorescent PCR amplification, wherein the reaction conditions are as follows: circulating for 1 time at 37 ℃ for 2min and 94 ℃ for 2 min; multiplying by 15s at 93 ℃ and 60s at 60 ℃, and circulating for 40 times; single-point fluorescence detection is carried out at 60 ℃, and a fluorescence signal is collected;
(6) for IS6110 sequence, phoE gene, OXA51 gene and ompP6 gene, FAM channel on fluorescent PCR instrument IS used for detection, for hlyA gene, toxA gene, ydiJ gene and ureR gene, ROX channel on fluorescent PCR instrument IS used for detection, for int gene, JOE channel on fluorescent PCR instrument IS used for detection, and the genotype of detection site IS determined by data collected by fluorescent quantitative PCR instrument.
Therefore, the application establishes a multiple fluorescence quantitative PCR reaction system with good specificity and sensitivity, 8 target genes are amplified in 4 PCR tubes, whether a sample contains mycobacterium tuberculosis, listeria monocytogenes, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, escherichia coli, haemophilus influenzae and proteus mirabilis can be determined through one-time detection, and a basis is provided for diagnosing the state of an illness.
In addition, the application uses real-time fluorescence quantitative PCR to detect mycobacterium tuberculosis, listeria monocytogenes, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, escherichia coli, haemophilus influenzae and proteus mirabilis, compared with the conventional clinical detection method, the detection period is greatly shortened, the efficiency and the detection flux are improved, the detection requirement is met, and the method has higher specificity, sensitivity and accuracy.
For a better understanding of the present disclosure, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings. It should be understood that these examples are for further illustration only and are not intended to limit the scope of the present application. In addition, after reading the contents of the present application, some insubstantial changes or modifications of the present application will still fall within the scope of the present application.
The experimental procedures in the following examples are, unless otherwise specified, routine in the art.
Example 1
This example provides the composition of an exemplary kit.
Specifically, the exemplary kit includes reagent bottles or tubes each containing the following components, and a packaging box collectively packaging these reagent bottles or tubes.
1) Nucleic acid extraction solution: 2% (M/V) Chelex-100, 1% (V/V) Tris-HCl, 1M, pH9.0;
2) PCR reaction enzyme system: 5U/mu L Taq DNA polymerase and 2U/mu L uracil-N-glycosylase (UNG enzyme) are mixed according to the volume ratio of 3: 1;
3) internal standard: a plasmid containing an internal standard int gene fragment;
4) negative control: double distilled water purified by a Millipore water purifier;
5) positive control: t-vector plasmid carrying target gene (IS6110) for detecting mycobacterium tuberculosis, T-vector plasmid carrying target gene (hlyA) for detecting listeria monocytogenes, T-vector plasmid carrying target gene (phoE) for detecting klebsiella pneumoniae, T-vector plasmid carrying target gene (toxA) for detecting pseudomonas aeruginosa, T-vector plasmid carrying target gene (OXA51) for detecting acinetobacter baumannii, T-vector plasmid carrying target gene (ydiJ) for detecting escherichia coli, T-vector plasmid carrying target gene (ompP6) for detecting haemophilus influenzae, T-vector plasmid carrying target gene (ureR) for detecting proteus mirabilis or engineering bacterium escherichia coli containing the above plasmids, and the bacterium concentration serving as a control bacterium IS 105CFU/mL；
6) First primer probe mixture: the kit consists of deoxyribonucleoside triphosphate dN (U) TP, a primer (shown as SEQ ID NO.1 and SEQ ID NO.2) of a mycobacterium tuberculosis target sequence (IS6110), a fluorescence labeling probe (shown as SEQ ID NO.3, a fluorescence reporter FAM IS labeled at the 5 'end, a quenching fluorescent group BHQ1 IS labeled at the 3' end), a primer (shown as SEQ ID NO.4 and SEQ ID NO.5) of a listeria monocytogenes target gene (hlyA), a fluorescence labeling probe (shown as SEQ ID NO.6, a fluorescence reporter ROX IS labeled at the 5 'end, a quenching fluorescent group BHQ2 IS labeled at the 3' end), a forward and reverse primer of an internal standard int gene and a fluorescence labeling probe, and the specific contents are as follows:
7) second primer probe mixture: composed of deoxyribonucleoside triphosphate dN (U) TP, a primer (shown as SEQ ID NO.7 and SEQ ID NO.8) of a Klebsiella pneumoniae target gene (phoE), a fluorescence labeling probe (shown as SEQ ID NO.9, a fluorescence reporter group FAM is labeled at the 5 'end, a fluorescence quenching group BHQ1 is labeled at the 3' end), a primer (shown as SEQ ID NO.10 and SEQ ID NO.11) of a pseudomonas aeruginosa target gene (toxA), a fluorescence labeling probe (shown as SEQ ID NO.12, a fluorescence reporter group ROX is labeled at the 5 'end, a fluorescence quenching group BHQ2 is labeled at the 3' end), a primer of an internal standard int gene and a fluorescence labeling probe, and the specific content is as follows:
8) third primer probe mixture: the kit consists of deoxyribonucleoside triphosphate dN (U) TP, a primer (shown as SEQ ID NO.13 and SEQ ID NO.14) of an acinetobacter baumannii target gene (OXA51), a fluorescence labeling probe (shown as SEQ ID NO.15, a fluorescence reporter group FAM is labeled at the 5 'end, a fluorescence quenching group BHQ1 is labeled at the 3' end), a primer (shown as SEQ ID NO.16 and SEQ ID NO.17) of an escherichia coli target gene (ydiJ), a fluorescence labeling probe (shown as SEQ ID NO.18, a fluorescence reporter group ROX is labeled at the 5 'end, a fluorescence quenching group Q2 is labeled at the 3' end, a primer of an internal standard int gene and a fluorescence labeling probe, wherein the contents are as follows:
9. fourth primer probe mixture: the kit consists of deoxyribonucleoside triphosphate dN (U) TP, primers (shown as SEQ ID NO.19 and SEQ ID NO.20) of a haemophilus influenzae target gene (ompP6), a fluorescence labeling probe (shown as SEQ ID NO.21, a fluorescence reporter group FAM is labeled at the 5 'end, a fluorescence quenching group BHQ1 is labeled at the 3' end), primers (shown as SEQ ID NO.22 and SEQ ID NO.23) of a proteus mirabilis target gene (ureR), a fluorescence labeling probe (shown as SEQ ID NO.24, a fluorescence reporter group ROX is labeled at the 5 'end, a fluorescence quenching group Q2 is labeled at the 3' end), a primer of an internal standard gene and a fluorescence labeling probe, wherein the contents are as follows:
in the mixed solution, the primers of the internal standard int gene are shown as SEQ ID NO.25 and SEQ ID NO.26, the fluorescence labeling probe of the internal standard int gene is shown as SEQ ID NO.27, the 5 'end labeling fluorophore is JOE, and the 3' end labeling quencher group TAMRA.
Example 2
This example provides a method for establishing assays using the exemplary kit of example 1.
Specifically, the method comprises the following steps:
1) preparing a reference substance: respectively placing 50 μ L of positive control and negative control in 1.5mL (or 0.5mL) centrifuge tube (shaking for 10s after the frozen reagent is melted), respectively adding 50 μ L of nucleic acid extract, mixing, keeping the temperature at 98 deg.C for 10min, centrifuging at 12,000rpm for 5min, and collecting 2 μ L of supernatant for PCR reaction.
2) Preparing a reaction system mixed solution: 16 mul of each primer probe mixed solution is mixed with 0.2 mul of PCR reaction enzyme system, evenly mixed by oscillation for a plurality of seconds, and evenly mixed by centrifugation at 3000rpm for a plurality of seconds.
3) And (3) PCR amplification: adding 2 mul of the treated supernatant of the negative control and the positive control into the PCR tube of the mixed solution of each reaction system prepared in the step 2), adding 2 mul of the internal standard into the PCR tube of the negative control and the PCR tube of the positive control, covering the tube cover, and immediately carrying out the fluorescence PCR amplification reaction.
The reaction procedure was as follows: circulating for 1 time at 37 ℃ for 2min and 94 ℃ for 2 min; multiplying by 15s at 93 ℃ and 60s at 60 ℃, and circulating for 40 times; single-point fluorescence detection is carried out at 60 ℃ in a reaction system of 20 mu L.
Fluorescence channel detection selection: IS6110, phoE, OXA51 and ompP6 genes select FAM channels, hlyA, toxA, ydiJ and ureR genes select ROX channels, and internal standard int genes select JOE channels.
The amplification results of the positive control are shown in FIGS. 1 to 4.
4) And (4) judging a result: after the detection is finished, the fluorescence signals of 6-15 cycles are taken for the baseline adjustment, and the threshold value is set according to the principle that the threshold value line just exceeds the highest point of the fluorescence curve detected by the negative control. Judging the detection result according to the CT value of the target gene amplification result, if the CT value is less than or equal to 35, indicating that the detection sample is lower than the detection limit, and reporting as negative; if the CT value is less than or equal to 35, the detection result is reported as positive; the CT display is a gray scale area between 35 and 40.
Example 3
This example provides specific experiments performed on the kits and methods of the present application.
1) Experimental Material
The experimental materials used in the following experiments and their sources are shown below:
| numbering | Pathogen species | Origin of origin |
| 1 | Enterococcus faecalis | Zhejiang Ningbo city disease control center |
| 2 | Enterococcus faecium | Zhejiang Ningbo city disease control center |
| 3 | Diphtheria bacillus | Zhejiang Ningbo city disease control center |
| 4 | Neisseria meningitidis | Zhejiang Ningbo city disease control center |
| 5 | Streptococcus sanguis | Zhejiang Ningbo city disease control center |
| 6 | Candida albicans | Zhejiang Ningbo city disease control center |
| 7 | Acid-producing Klebsiella sp | Zhejiang Ningbo city disease control center |
| 8 | Burkholderia cepacia | Zhejiang Ningbo city disease control center |
| 9 | Staphylococcus aureus | Zhejiang Ningbo city |
| 10 | Staphylococcus epidermidis | Zhejiang Ningbo city disease control center |
| 11 | Positive control | See example 1 |
| 12 | Negative control | See example 1 |
2) Experimental methods
The kit in example 1 is used, and the experimental method established in example 2 is used to detect the 10 pathogenic bacteria respectively, so as to analyze and evaluate the specificity of the kit.
3) Results of the experiment
The 10 common pathogenic bacteria (enterococcus faecalis, enterococcus faecium, corynebacterium diphtheriae, neisseria meningitidis, streptococcus sanguis, candida albicans, klebsiella oxytoca, burkholderia cepacia, staphylococcus aureus, staphylococcus epidermidis) were detected using the kit described in example 1, and all the results were negative (see table 1).
Table 1 test results of the specificity of the kit of the present application
As can be seen, the above results indicate that the detection specificity of the kit and the method of the application is good.
Example 4
This example provides a sensitivity assay performed on the kits and methods of the present application.
1) Experimental Material
4 concentration gradients (5X 10) were established, respectively5copy/mL, 5X 104copy/mL, 5X 103copy/mL, 5X 102copy/mL) as a sensitivity reference for the kit.
2) Experimental methods
Plasmid positive reference products with 4 concentration gradients of IS6110, hlyA, phoE, toxA, OXA51, ydiJ, ompP6 and ureR genes are respectively detected, and the sensitivity of the kit for detecting the plasmid concentration (copy/mL) IS determined.
The specific method comprises the following steps: measuring OD (260nm) of plasmid reference, converting into copy/mL unit, performing gradient dilution, and selecting 4 concentration gradients of 5 × 105copy/mL, 5X 104copy/mL, 5X 103copy/mL, 5X 102copy/mL was used as reference.
The above reference was subjected to sensitivity test using the kit of example 1. And (4) repeatedly detecting each concentration reference substance for 20 times, counting the positive detection times of each concentration, and determining the lowest detection limit of the product.
3) Results of the experiment
The kit in the embodiment 1 is used for detecting plasmid positive reference substances with different concentrations by adopting the experimental method established in the embodiment 2, and the kit can stably detect 5X 104copies/mL plasmid positive reference, results are shown in Table 2.
TABLE 2 number of plasmid positive reference detections at each concentration
As can be seen, according to the results of the sensitivity test, the kit of the present application has a minimum detection limit of 5X 10 determined according to the plasmid copy number concentration4copy/mL.
Example 5
This example uses 3 batches of the kit of example 1 and the protocol set up in example 2 for plasmid positive references (5X 10) for IS6110, hlyA, phoE, toxA, OXA51, ydiJ, ompP6 and ureR genes, respectively (5X 10)4copy/mL) were repeated.
1) Experimental Material
The 5X 10 in example 4 was used4The precision of the kit is researched by taking a copy/mL plasmid positive reference as an experimental object.
2) Experimental methods
(1) Replicate experiment in batch: using plasmid positive reference substances prepared at the same time, and repeatedly detecting each reference substance 3 times according to the method established in example 2;
(2) batch-to-batch repeat experiments: plasmid positive references prepared in 3 batches (P1, P2, P3) at different times were used, and each reference was tested in 3 replicates according to the method established in example 2.
(3) Results of the experiment
The results are shown in tables 3 and 4, and the variation coefficient of precision (CV%) was 5% or less in both the batch and the batch.
TABLE 3 results of the in-batch reproducibility study
TABLE 4 results of the batch to batch reproducibility study
| Gene | Mean value (C)T) | SD | CV％ |
| IS6110 | 26.4001 | 0.1328 | 0.50 |
| hlyA | 26.3534 | 0.1877 | 0.71 |
| phoE | 25.7461 | 0.3431 | 1.33 |
| toxA | 25.8849 | 0.1327 | 0.51 |
| OXA51 | 26.5391 | 0.105 | 0.40 |
| ydiJ | 26.2736 | 0.204 | 0.78 |
| ompP6 | 25.566 | 0.0831 | 0.32 |
| ureR | 26.3865 | 0.2914 | 1.10 |
Example 6
This example provides for the detection of clinical specimens using the kits and methods of the present application.
For 30 clinical samples collected from State people's institute of Ningbo, Zhejiang province, the samples included: 1 example of Mycobacterium tuberculosis, 1 example of Listeria monocytogenes, 4 examples of Klebsiella pneumoniae, 2 examples of Pseudomonas aeruginosa, 2 examples of Acinetobacter baumannii, 7 examples of Escherichia coli, 1 example of Haemophilus influenzae, and 1 example of Proteus mirabilis.
The test results using the kit of the present application were compared with the results of clinical bacterial culture identification and drug susceptibility tests, and the results are shown in table 5.
TABLE 5 results of compliance experiments
As can be seen, the detection results show that the detection results of the kit are completely consistent with the clinical bacteria culture identification test results.
The above description is not intended to limit the present application, nor is the present application limited to the above examples. Those skilled in the art should also realize that such changes, modifications, additions and substitutions are within the spirit and scope of the present application.
Claims (10)
1. A primer and/or probe composition for detecting a bacillus that primes a bloodstream infection, comprising a primer and/or probe selected from the group consisting of primers and/or probes for detecting one or more of the following targets: specific insertion sequences (IS6110 sequence) of mycobacterium tuberculosis complex, listeria monocytogenes hemolysin gene (hlyA gene), klebsiella pneumoniae outer membrane phosphoprotein gene (phoE gene), pseudomonas aeruginosa ETA protein coding gene (toxA gene), acinetobacter baumannii oxacillin gene (OXA51 gene), escherichia coli putative protein gene (ydiJ gene), haemophilus influenzae outer membrane protein P6 gene (ompP6 gene), and proteus mirabilis urease synthesis regulatory factor gene (ureR gene).
2. The primer and/or probe composition of claim 1, comprising primers and/or probes for detecting the IS6110 sequence and/or the hlyA gene, respectively;
optionally, it comprises primers and/or probes for detecting the phoE gene and/or the toxA gene, respectively;
optionally, it comprises primers and/or probes for detecting the OXA51 gene and/or the ydiJ gene, respectively; and/or
Optionally, it comprises primers and/or probes for detecting the ompP6 gene and/or the ureR gene, respectively.
3. The primer and/or probe composition according to claim 1 or 2, wherein the primer sequence for detecting the IS6110 sequence IS shown as SEQ ID No.1 and SEQ ID No.2, and the probe sequence IS shown as SEQ ID No. 3;
optionally, the primer sequence for detecting the hlyA gene is shown as SEQ ID NO.4 and SEQ ID NO.5, and the probe sequence is shown as SEQ ID NO. 6;
optionally, the primer sequence for detecting the phoE gene is shown as SEQ ID NO.7 and SEQ ID NO.8, and the probe sequence is shown as SEQ ID NO. 9;
optionally, the primer sequence for detecting the toxA gene is shown as SEQ ID NO.10 and SEQ ID NO.11, and the probe sequence is shown as SEQ ID NO. 12;
optionally, the primer sequence for detecting the OXA51 gene is shown as SEQ ID NO.13 and SEQ ID NO.14, and the probe sequence is shown as SEQ ID NO. 15;
optionally, the primer sequence for detecting the ydiJ gene is shown as SEQ ID NO.16 and SEQ ID NO.17, and the probe sequence is shown as SEQ ID NO. 18;
optionally, the primer sequence for detecting the ompP6 gene is shown as SEQ ID NO.19 and SEQ ID number 20, and the probe sequence is shown as SEQ ID NO. 21; and/or
Optionally, the primer sequence for detecting the ureR gene is shown as SEQ ID NO.22 and SEQ ID NO.23, and the probe sequence is shown as SEQ ID NO. 24.
4. The primer and/or probe composition of any one of claims 1 to 3, wherein said probe sequence is labeled with a fluorescent reporter group and a quencher group, respectively;
optionally, the fluorescent reporter group is selected from FAM, ROX, JOE, HEX or TET; and/or the quenching group is selected from BHQ1, BHQ2 or TAMRA.
5. The primer and/or probe composition of any one of claims 1 to 4, further comprising deoxyribonucleoside triphosphates dN (U) TP.
6. Use of a primer and/or probe composition according to any one of claims 1 to 5 in the manufacture of a reagent or kit for the detection of bacilli that cause a bloodstream infection.
7. A reagent or kit for detecting a bacillus causing a bloodstream infection comprising the primer and/or probe composition of any one of claims 1 to 5.
8. The reagent or kit of claim 7, further comprising primers and/or probes for detecting internal standard genes;
optionally, the internal reference gene is an int gene;
optionally, the primer sequence for detecting the int gene is shown as SEQ ID NO.25 and SEQ ID NO.26, and the probe sequence is shown as SEQ ID NO. 27.
9. The reagent or kit of claim 7 or 8, further comprising one or more of: nucleic acid extract, PCR reaction enzyme system, negative reference substance or positive reference substance;
optionally, the nucleic acid extract comprises: 2% (M/V) Chelex-100 and 1% (V/V) Tris-HCl;
optionally, the PCR reaction enzyme system comprises: 5U/muL Taq DNA polymerase and 2U/muL uracil-N-glycosylase;
optionally, the negative control is purified double distilled water;
optionally, the positive control is a T-vector plasmid carrying the detected target or an engineered escherichia coli bacterium containing the T-vector plasmid carrying the detected target.
10. A method for detecting a bacillus causing a bloodstream infection comprising detecting a biological sample using the primer and/or probe composition of any one of claims 1 to 5 or the reagent or kit of any one of claims 7 to 9.
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| CN111187848A (en) * | 2020-01-22 | 2020-05-22 | 北京卓诚惠生生物科技股份有限公司 | Nucleic acid reagent, kit, system and method for detecting blood stream infection pathogenic bacteria |
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