Screening and application of nocardia gilvus marker

Abstract

The application provides a set of biomarkers for identifying and detecting Nocardia, wherein the biomarkers are one or more of NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299 and C5B73_13220, the biomarkers are nucleic acid sequences or amino acid sequences, the nucleic acid sequences of the biomarkers NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299 and C5B73_13220 are shown in SEQ ID NO. 1-5, and the amino acid sequences are shown in SEQ ID NO. 6-10. By identifying the molecular markers, the Gelsenkirchen Nocardia can be accurately identified and detected, and the process is simpler, faster, more accurate and lower in cost, so that the differential diagnosis of diseases caused by the Gelsenkirchen Nocardia is at least partially achieved, and the cure rate and prognosis are improved.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to the screening and application of Nocardia glyphosate biomarkers. Background Technology

[0002] Nocardia is a Gram-positive bacterium with branching hyphae. Clinically, Nocardia infections are relatively rare, usually involving acute or chronic lung infections. Nocardia is widely distributed in soil, decaying vegetation, animal feces, freshwater, and seawater. It was first isolated from cattle by Nocard in 1888. It primarily spreads through the respiratory tract, causing lung abscesses in humans and animals. It can also cause serious opportunistic infections of human organs (including the lungs, skin, and central nervous system) through wounds or blood transmission. Susceptible populations are mainly immunocompromised individuals (e.g., those with HIV / AIDS, organ transplants, or diabetes), but occasionally it can infect immunocompetent individuals. Although identifying Nocardia at the species level is crucial for assessing pathogenicity, predicting antibiotic susceptibility, and prognosis, the classification of Nocardia remains controversial. Clinically, pathogenic Nocardia species in humans include *Nocardia melioides*, *Nocardia asteroides*, *Nocardia brasiliensis*, and *Nocardia gailsenkirchen*. Different species of Nocardia exhibit significant differences in geographical distribution, biochemical characteristics, phenotypic characteristics, and drug susceptibility.

[0003] Nocardia cyriacigeorgica is a common clinically relevant pathogen classified from Nocardia asteroides and is one of the important pathogens among Nocardia species. First reported in 2001, the incidence of Nocardia cyriacigeorgica infection has been increasing in recent years due to the rise in immunocompromised patients. In North America, this species is the most common Nocardia species causing nocardiac infection. Furthermore, cases of Nocardia cyriacigeorgica infection have been reported in Canada, France, Greece, the Netherlands, Turkey, Iran, and China, demonstrating a global distribution. Nocardia cyriacigeorgica is susceptible to cephalosporins, amikacin, linezolid, and imipenem. In addition, for patients with severe cases, most clinicians recommend combination therapy with trimethoprim (TMP) and sulfamethoxazole (SMX). However, in 2018, there were reports of TMP-SMX resistant Nocardia gailsenigcheni, which requires our attention.

[0004] Nocardiac infection shares similarities with common bacterial and fungal infections, with clinical manifestations primarily including nonspecific symptoms such as cough, fever, and chest pain. Traditional diagnostic methods often result in weakly acid-fast smears, easily confused with Mycobacterium tuberculosis, leading to confusion in the identification of these pathogens and potential misdiagnosis. This can delay optimal treatment and result in high mortality rates. Therefore, the possibility of Nocardia infection should be considered in patients with pulmonary infections who have failed empirical antibacterial, antifungal, or antituberculosis treatment and possess the aforementioned risk factors. Furthermore, different Nocardia species exhibit different antimicrobial susceptibility patterns. Species-level identification of Nocardia is crucial for assessing antibiotic susceptibility and pathogenicity. However, the genome sequences of Nocardia species are highly similar, making biochemical identification methods insufficient for species differentiation. For Nocardia gelsenkirchen, a common pathogen, the gold standard for identification is 16S rDNA sequencing, a time-consuming and complex method that presents a challenge for conventional laboratories.

[0005] Therefore, it is necessary to study a simple, rapid, and accurate biomarker for identifying and detecting Nocardia cyriacigeorgica, so as to enable early diagnosis and timely and correct treatment of diseases caused by Nocardia cyriacigeorgica, thereby improving the cure rate and prognosis. Summary of the Invention

[0006] In a first aspect, the present invention provides a set of biomarkers for identifying and detecting Nocardia, wherein the biomarkers are one or more nucleic acids or amino acids selected from NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299, and C5B73_13220.

[0007] Furthermore, the nucleic acid sequences of the biomarkers NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299, and C5B73_13220 are shown in SEQ ID NO.1 to 5, respectively.

[0008] Furthermore, the amino acid sequences of the biomarkers NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299, and C5B73_13220 are shown in SEQ ID NO.6 to 10.

[0009] Furthermore, the Nocardia is Nocardia gailans.

[0010] Secondly, the present invention provides a set of primers for identifying and detecting Nocardia biomarkers, wherein the biomarkers are NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299, and C5B73_13220. The primer sequences of the biomarkers are shown in SEQ ID NO. 11 to 20, respectively. Specifically, the upstream and downstream primer sequences of NCTC10797_02287 are shown in SEQ ID NO. 11 to 12; the upstream and downstream primer sequences of NCTC10797_01760 are shown in SEQ ID NO. 13 to 14; the upstream and downstream primer sequences of NCTC10797_05842 are shown in SEQ ID NO. 15 to 16; and the upstream and downstream primer sequences of NOCYR_2299 are shown in SEQ ID NO. 13 to 20. As shown in NO.17-18, the upstream and downstream primer sequences of C5B73_13220 are shown in SEQ ID NO:19-20.

[0011] Thirdly, the present invention provides the application of the biomarker described in the first aspect in the preparation of a kit for the differential diagnosis of Nocardia, wherein the biomarker is one or more nucleic acid sequences or amino acids selected from NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299, and C5B73_13220.

[0012] Furthermore, the nucleic acid sequences of the biomarkers NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299, and C5B73_13220 are shown in SEQ ID NO.1 to 5, respectively.

[0013] Furthermore, the amino acid sequences of the biomarkers NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299, and C5B73_13220 are shown in SEQ ID NO.6 to 10.

[0014] Furthermore, if the patient's biological sample contains one or more of the aforementioned biomarkers NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299, and C5B73_13220, then the patient's biological sample is determined to contain Nocardia.

[0015] Furthermore, the patient's biological samples were selected from blood, pleural fluid, or sputum.

[0016] Furthermore, the Nocardia is Nocardia gailans.

[0017] Fourthly, the present invention provides a kit for detecting and identifying biomarkers of Nocardia, the kit containing reagents for detecting the biomarkers, wherein the biomarkers are one or more nucleic acids or amino acids selected from NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299, and C5B73_13220.

[0018] Furthermore, the nucleic acid sequences of the biomarkers NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299, and C5B73_13220 are shown in SEQ ID NO. 1 to 5; and the amino acid sequences of the biomarkers NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299, and C5B73_13220 are shown in SEQ ID NO. 6 to 10.

[0019] Furthermore, the detection reagent for the biomarker contains primers as shown in SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18 and SEQ ID NO.19-20.

[0020] Fifthly, the present invention provides a method for screening biomarkers for nocardiac disease, the method comprising the following steps:

[0021] S1. Obtain samples to obtain Nocardia strains with complete genomes, which will serve as the discovery and validation groups;

[0022] S2. Annotate the genomes of the above samples;

[0023] S3. Pan-genome analysis was performed on the gene sequences annotated in S2 to identify specific genes;

[0024] S4. Selected specific genes for diagnosing Nocardia are used to design primers based on their gene sequences. The obtained primers are then subjected to sequence alignment analysis in a database to ultimately obtain specific biomarkers.

[0025] Furthermore, the genomic data annotation includes core genes, non-core genes, and unique genes.

[0026] Furthermore, the core gene refers to a gene that exists in all plant and animal strains or strains; the non-core gene refers to a gene that exists in one or more plant and animal strains or strains; and the unique gene refers to a gene that exists only in one strain.

[0027] Furthermore, the pan-genome analysis involves comparing the obtained unique genes with the gene sequences annotated in the validation group.

[0028] Furthermore, Prokka software was used for genome data annotation, BPGA software for pan-genome analysis, and BLAT software for gene sequence alignment.

[0029] Furthermore, the gene sequence alignment is performed using a 50% identity threshold and a 50% coverage rate as standards for gene homology analysis and comparative gene analysis.

[0030] Further, the biomarkers are NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299, and C5B73_13220. The primer sequences for these biomarkers are shown in SEQ ID NO. 11–20, respectively. Specifically, the upstream and downstream primer sequences for NCTC10797_02287 are shown in SEQ ID NO. 11–12; the upstream and downstream primer sequences for NCTC10797_01760 are shown in SEQ ID NO. 13–14; the upstream and downstream primer sequences for NCTC10797_05842 are shown in SEQ ID NO. 15–16; the upstream and downstream primer sequences for NOCYR_2299 are shown in SEQ ID NO. 17–18; and the upstream and downstream primer sequences for C5B73_13220 are shown in SEQ ID NO. 11–20. NO:19~20 shown.

[0031] Beneficial effects:

[0032] The present invention provides Nocardia gailansii with specific molecular markers that distinguish it from other Nocardia species, including NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299, and C5B73_13220. By identifying these molecular markers, Nocardia gailansii can be accurately identified and detected, thereby at least partially achieving the differential diagnosis of diseases caused by Nocardia gailansii, improving its cure rate and prognosis. Attached Figure Description

[0033] Figure 1 Gene accumulation curves for the pangenome and the core genome;

[0034] Figure 2 Primer pair verification and detection results based on NCTC10797_02287;

[0035] Figure 3 Primer pair verification and detection results based on NCTC10797_01760;

[0036] Figure 4 Primer pair verification and detection results based on NCTC10797_05842;

[0037] Figure 5 Primer pair verification and detection results based on NOCYR_2299;

[0038] Figure 6 Primer pair verification and detection results based on C5B73_13220. Detailed Implementation

[0039] The following provides a detailed description of specific embodiments of this disclosure. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of this disclosure.

[0040] Unless otherwise specified, the raw materials, reagents, instruments and equipment involved in the embodiments of this invention can all be obtained by purchase.

[0041] Example 1: Screening of biomarkers for Nocardia gailans.

[0042] Thirty-three Nocardia strains with complete genomes were collected as the discovery group. These included 16 subspecies, among other common Nocardia strains: 3 *Nocardia gailans*, 4 *Nocardia dermatophytes*, 5 *Nocardia asteroides*, and 3 *Nocardia brasiliensis*.

[0043] In addition, 297 strains with original sequencing reads were collected as the first validation group. These included common Nocardia strains such as 32 strains of Nocardia gailans, 40 strains of Nocardia dermatophytes, 3 strains of Nocardia asteroides, and 7 strains of Nocardia brasiliensis.

[0044] The Prokka prokaryotic genome rapid annotation software was used to perform rapid and efficient functional annotation of the genomic data of the discovery and validation groups; the BPGA software was used to perform pan-genome analysis of the annotated gene sequences of the discovery group (see...). Figure 1Gene clustering was performed on common genes for each species to identify core genes, non-core genes, and unique genes for each species. The unique genes were compared with the gene sequences annotated in the validation group using BLAT software. Gene homology analysis and comparative gene analysis were performed using a 50% identity threshold and a 50% coverage rate as criteria. A total of 44 specific genes were identified as candidate biomarkers for the diagnosis of Nocardia gailjan.

[0045] After selecting 44 specific genes as candidate biomarkers, primers were designed based on the gene sequences using Primer Premier 5.0 software. The obtained primers were then subjected to sequence alignment analysis in the NCBI database (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to exclude specific gene sequences with high homology to other species. At the same time, non-specific matches between the designed primers and sequences of other species were eliminated. Finally, 5 relatively specific biomarkers were obtained (the nucleic acid sequences of the biomarkers are shown in Table 1, and the amino acid sequences are shown in Table 2). The optimized specific amplification primers are shown in Table 3.

[0046] Table 1 Nucleic acid sequences of biomarkers

[0047]

[0048]

[0049]

[0050]

[0051] Table 2 Amino acid sequences of biomarkers

[0052]

[0053]

[0054] Table 3. Primer sequence listing for biomarkers

[0055]

[0056] Example 2: Detection effect of biomarkers on Nocardia

[0057] Twenty-four clinical Nocardia strains and other strains were collected, and genomic DNA was extracted from these strains using a Qiagen DNA extraction kit according to the manufacturer's instructions. The concentration and purity of the genomic DNA were determined using a UV spectrophotometer. All genomic DNA samples were aliquoted and stored at -20°C for later use.

[0058] The PCR amplification reaction program was 94℃ for 5 min, 94℃ for 30 s, 68℃ for 30 s, 72℃ for 30 s (40 cycles), and 72℃ for 2 min to obtain the PCR amplification product. (The primer pairs for the specific molecular markers of Nocardia gailans may include at least one pair of the primers shown in SEQ ID NO. 11-12, SEQ ID NO. 13-14, SEQ ID NO. 15-16, SEQ ID NO. 17-18, and SEQ ID NO. 19-20, wherein the sequences of the above primer pairs are shown in Table 3).

[0059] Prepare a 1.5% agarose gel using standard methods. Add 5 μL of sample amplification product to each well, and leave one well empty to add 5 μL of DNA marker. Electrophoresis is performed at 120 V for approximately 30 minutes. Observe the electrophoresis results under UV light. The order of bacterial strains added to the sample wells and the results for agarose gel electrophoresis are shown in Table 4. Electrophoresis results are shown in the figure (see Table 4). Figure 2-6 ).

[0060] Table 4. Strain order and results of 24 clinical Nocardia strains and other strains in the sample wells.

[0061] bacteria strain source quantity Sample well PCR results Marker - - 1 - Nocardia glyphosate of Gelsenkirchen Clinical isolates 8 2-9 Positive Nocardia anthrax Clinical isolates 4 10-13 Negative Nocardia guinea pig otitis media Clinical isolates 1 14 Negative Nocardia abscess Clinical isolates 1 15 Negative Wallace Nocardia Clinical isolates 1 16 Negative Beijing Nocardia Clinical isolates 1 17 Negative Nocardia cinerea Clinical isolates 1 18 Negative Nocardia pustulosa Clinical isolates 1 19 Negative Tsukamura fungi Clinical isolates 1 10 Negative genus Gordonia Clinical isolates 1 21 Negative Mycobacterium tuberculosis H37Rv standard strain 1 22 Negative Mycobacterium tectorum Standard strain ATCC35750 1 23 Negative Mycobacterium abscessus Standard strain ATCC19977 1 24 Negative Mycobacterium avium Standard strain ATCC25291 1 25 Negative

[0062] From Table 4 and Figure 2-6 It is understood that the biomarker provided by the present invention can realize the detection of Nocardia glycerides in Gelsenkirchen, and the process is simpler, faster, more accurate and lower in cost.

Claims

1. Use of a detection reagent of a biomarker in the preparation of a kit for differential diagnosis of Nocardia, wherein the Nocardia is Nocardia gilvus; the biomarker is a nucleic acid or an amino acid of NCTC10797_02287; the nucleic acid sequence of the biomarker NCTC10797_02287 is shown as SEQ ID NO. 1; the amino acid sequence of the biomarker NCTC10797_02287 is shown as SEQ ID NO. 6; the biomarker is NCTC10797_02287, and the upstream and downstream primer sequences of the NCTC10797_02287 are shown as SEQ ID NO. 11-12.

2. Use according to claim 1, wherein The biological sample of the patient is selected from blood, pleural tissue fluid or sputum.

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