CN118345186A - Voriconazole drug resistance detection method for Marneffei basket bacteria - Google Patents
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- 206010059866 Drug resistance Diseases 0.000 title claims abstract description 33
- BCEHBSKCWLPMDN-MGPLVRAMSA-N voriconazole Chemical compound C1([C@H](C)[C@](O)(CN2N=CN=C2)C=2C(=CC(F)=CC=2)F)=NC=NC=C1F BCEHBSKCWLPMDN-MGPLVRAMSA-N 0.000 title claims abstract description 19
- 229960004740 voriconazole Drugs 0.000 title claims abstract description 19
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- 238000001514 detection method Methods 0.000 title claims abstract description 11
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
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Abstract
The invention discloses a voriconazole drug resistance detection method of a Marneffei basket fungus, which comprises the following steps: the method comprises the following steps: detecting a target enzyme gene cyp51B coding region of the Marneffei basket, wherein the nucleotide sequence of the target enzyme gene cyp51B coding region is shown as SEQ NO 1; if at least one of 1581, 1583 and 1587 of the coding region of the target enzyme gene cyp51B is mutated, the generation of drug resistance of the Markife basket bacteria is judged. According to the invention, through the change of the SNP locus related to the drug resistance gene and the change of the gene expression quantity as the early prediction index of the drug resistance of the voriconazole of the Langmuir marneffei, the early detection of the drug resistance of the Langmuir marneffei can be realized.
Description
Technical field:
the invention belongs to the field of medicines, and particularly relates to a voriconazole drug resistance detection method of a basket-shaped marneffei.
The background technology is as follows:
Marneffe basket (Talaromyces marneffei) is the only temperature dependent biphasic fungus in the genus Pentium, and its caused Marneffe basket disease is endemic in southeast Asia, especially Guangxi, and mainly infects immunocompromised persons, especially AIDS patients. 19% of AIDS patients in Guangxi are statistically combined with a panifenesin infection, the second most opportunistic infection next to tuberculosis (Tang Xiuwen, zhang Jing. Guangxi medical, 2010 (07)). Systemic marneffei basket disease causes severe damage to various tissues and organs, and if not the most effective treatment mortality rate (more than 80%) is extremely high, it is an important infective element leading to the death of AIDS in I district (Liyan X, CHANGMING L, xianyi Z, luxia W, suisheng X.mycopathologia 2004;158 (2): 151-5.)
Amphotericin B (AMB) is currently the first choice for clinical treatment of this disease. However, patients often cannot tolerate AMB treatment due to the large side effects of AMB; however, since voriconazole (Voriconazole, VOC) has a small side effect, it is used as a substitute for (Hoenigl M,almanton-Garcia J,Walsh TJ,Nucci M,Neoh CF,Jenks JD,et al.Lancet InfectDis 2021,21(8):e246-e257). which cannot tolerate the toxic side effect of amphotericin B but the bioavailability of VOC for the treatment of Marneffei basket disease is unstable, and it has been found in recent years that voriconazole resistant strains (MIC. Gtoreq.1. Mu.g/ml) are useful.
The research on the drug resistance mechanism of other fungi triazole drugs shows that the specific mechanism of drug resistance of different pathogenic fungi is not the same. Mainly focused on two aspects: 1. the target enzyme of azole drugs, namely lanosterol 14-alpha-demethylase (14 DH), is overproduced and the gene mutation, so that the reduced affinity of the azole drugs for the enzyme is an important reason for the drug resistance of fungi to the azole drugs. The 14-alpha-demethylase encoding gene ERG11 mRNA is found to be over-expressed in azole-resistant candida albicans, candida glabrata and candida glabrata, and the point mutation of the ERG11 gene in aspergillus fumigatus is involved in the occurrence of azole-resistant. 2. Enhancement of drug efflux pump action is a major cause of drug resistance, which is often accompanied by upregulation of the gene encoding the efflux pump. Over-expression of such genes can reduce drug penetration and decrease intracellular azole concentration, resulting in azole resistance. At least 2 efflux pump systems play a role in the drug resistance process: MF family (major facilitator, encoded by MDR gene) and ABC transporter belonging to the ABC (ATP-binding cassette) family (encoded by CDR gene). Both have multiple homologous genes in the fungal genome. However, only the CDR1 and CDR2 genes of candida albicans, cdCDR genes of candida dublinii, cgCDR and CgCDR genes of candida glabrata and CnCDR genes of cryptococcus neoformans are positively regulated and correlated with azole resistance. The MDR1 gene in the MF family gene of another efflux pump is related to drug resistance, and some clinical isolates of Voriconazole resistant to drug overexpress MDR1. Unlike CDR1, the strain that overexpresses MDR1 is resistant to voriconazole and not to other azoles. At present, research on the gene functions of the Marneffei basket is still in a preliminary stage, and the random sequence tag (SET) discovers that azole drug target enzyme genes cyp51B, ERG, multi-drug resistance genes CDR, PMFCZ and the like exist in the genome of the Marneffei basket, but the research is only performed on the listing of drug resistance genes, and the change of the expression quantity of the drug resistance genes and the change of the expression quantity of related drug efflux pump genes are not reported yet.
The invention comprises the following steps:
the invention aims to provide a voriconazole drug resistance detection method of a Marneffei basket fungus.
In order to solve the problems, the technical scheme of the invention is as follows:
the voriconazole drug resistance detection method of the Marneffei basket bacteria comprises the following steps: detecting a target enzyme gene cyp51B coding region of the Marneffei basket, wherein the nucleotide sequence of the target enzyme gene cyp51B coding region is shown as SEQ NO 1; if at least one of 1581, 1583 and 1587 of the coding region of the target enzyme gene cyp51B is mutated, the generation of drug resistance of the Markife basket bacteria is judged.
Further improvement, if at least one of the conditions of the 1581 th position of the encoding region of the target enzyme gene cyp51B from G to A, the 1583 rd position from T to C and the 1587 th position from G to A occurs, the generation of drug resistance of the Langmuir marneffei is judged.
The utility model provides a voriconazole drug resistance detection method of Marneffei basket, which is used for detecting the expression quantity of genes AtrF, mdr1 and PMFCZ of Marneffei basket without drug resistance and the expression quantity of genes AtrF, mdr1 and PMFCZ of Marneffei basket to be detected; if the expression quantity of the cause AtrF, mdr1, PMFCZ of the Markov basket bacteria to be detected is larger than the expression quantity of the gene AtrF, mdr1, PMFCZ of the mark flow basket bacteria without drug resistance, the mark flow basket bacteria to be detected has drug resistance, otherwise, the mark flow basket bacteria to be detected has no drug resistance.
Compared with the prior art, the invention has the following advantages:
The early detection of the drug resistance of the Marneffei basket bacteria can be realized by using the change of the SNP locus related to the drug resistance gene and the change of the gene expression quantity as the early prediction index of the drug resistance of the Marneffei basket bacteria voriconazole treatment.
The specific embodiment is as follows:
the selected fungi are Malifer basket fungus standard strain FRR2161 and voriconazole drug-resistant strain (MIC is more than or equal to 1), and the selected drug is voriconazole.
Firstly, the full length bidirectional sequencing of the coding region of the target enzyme gene cyp51B is respectively carried out on a standard strain and a drug-resistant strain, and the sequencing result is compared with the cyp51B sequence of the standard strain through BLAST, the sequence is shown as SEQ NO2, and the result is shown as follows: the encoding region of the gene cyp51B of strain 229341 has a G1581A mutation, see SEQ NO3, resulting in the substitution of glycine at position 439 of 14-DM with aspartic acid (G439D); the encoding region of the gene cyp51B of the strain 198039 has a T1583C mutation, see sequence SEQ NO4, which results in the substitution of tyrosine at position 439 of 14-DM with histidine (Y440H) sequence SEQ NO5; the coding region of the gene cyp51B of strain A1 has a G1587A mutation, see sequence SEQ NO6, resulting in the replacement of glycine at position 441 of 14-DM with aspartic acid (G441D), see sequence SEQ NO7.
The difference of the expression amounts of target enzyme genes cyp51B, efflux pump genes ABC, atrF, mdr1, MFS and PMFCZ of the standard strain FRR2161 and the drug-resistant strain is detected by RT-PCR by taking beta-action as an internal reference gene. The results were as follows:
Table 1 mutation site and amino acid substitution site of target enzyme cyp51B gene of each experimental strain
Table 2 relative expression levels of the genes cyp51B, ABC, atrF, mdr, MFS and FLC of the standard strain FRR2161 and the resistant strains 229341, 198039 and A1 of Marneffei basket.
Compared with a standard strain, the drug-resistant strain is compared with the standard strain through t test comparison of independent samples, the expression level of genes cyp51B, atrF, mdr and PMFCZ is increased, and the difference has statistical significance (P is less than 0.05); the difference of the expression quantity of the genes ABC and MFS has no statistical significance (P is more than 0.05).
The above embodiment is only one specific implementation of the present invention, and is not intended to limit the present invention, and any simple modification and substitution thereof are within the scope of the present invention.
Claims (3)
1. The voriconazole resistance detection method of the Marneffei basket is characterized by comprising the following steps of: detecting a target enzyme gene cyp51B coding region of the Marneffei basket, wherein the nucleotide sequence of the target enzyme gene cyp51B coding region is shown as SEQ NO 1; if at least one of 1581, 1583 and 1587 of the coding region of the target enzyme gene cyp51B is mutated, the generation of drug resistance of the Markife basket bacteria is judged.
2. The method for detecting voriconazole resistance of a pannix marneffei according to claim 1, wherein it is judged that the pannix marneffei has developed resistance when at least one of the three cases of mutation from G to a at 1581 st, mutation from T to C and mutation from G to a at 1587 st in the coding region of the target enzyme gene cyp51B occurs.
3. The voriconazole resistance detection method of the marneffei basket is characterized by detecting the expression quantity of genes AtrF, mdr1 and PMFCZ of the marneffei basket without drug resistance and the expression quantity of genes AtrF, mdr1 and PMFCZ of the marneffei basket to be detected; if the expression quantity of the cause AtrF, mdr1, PMFCZ of the Markov basket bacteria to be detected is larger than the expression quantity of the gene AtrF, mdr1, PMFCZ of the mark flow basket bacteria without drug resistance, the mark flow basket bacteria to be detected has drug resistance, otherwise, the mark flow basket bacteria to be detected has no drug resistance.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112255354A (en) * | 2020-09-11 | 2021-01-22 | 广州医科大学附属第一医院 | Characteristic substance for diagnosing Marneffei panuliasis and screening method and application thereof |
| CN114657242A (en) * | 2022-03-16 | 2022-06-24 | 广州医科大学附属第一医院 | Application of GPR33 gene in assessment of marneffei Talaromyces susceptible population |
| CN114717353A (en) * | 2022-03-14 | 2022-07-08 | 广州医科大学附属第一医院 | Biomarker for auxiliary diagnosis of HIV negative Marneffei panuliferus disease and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112255354A (en) * | 2020-09-11 | 2021-01-22 | 广州医科大学附属第一医院 | Characteristic substance for diagnosing Marneffei panuliasis and screening method and application thereof |
| CN114717353A (en) * | 2022-03-14 | 2022-07-08 | 广州医科大学附属第一医院 | Biomarker for auxiliary diagnosis of HIV negative Marneffei panuliferus disease and application thereof |
| CN114657242A (en) * | 2022-03-16 | 2022-06-24 | 广州医科大学附属第一医院 | Application of GPR33 gene in assessment of marneffei Talaromyces susceptible population |
Non-Patent Citations (1)
| Title |
|---|
| 姚婉玉等: "马尔尼菲蓝状菌氟康唑耐药基因的表达", 中国皮肤性病学杂志, vol. 31, no. 6, 30 June 2017 (2017-06-30), pages 612 - 615 * |
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