Kit and system for nocardia detection

Abstract

The application provides a biomarker for identifying and detecting common pathogenic Nocardia, the biomarker is F6W96_34950, the biomarker is a nucleic acid sequence or an amino acid sequence, the nucleic acid sequence of the biomarker F6W96_34950 is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2. Through identification of the biomarkers, the application can accurately identify and detect Nocardia, and the process is simpler, faster, more accurate and lower in cost, so that at least partial differential diagnosis of diseases caused by Nocardia is realized, and the cure rate and prognosis are improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to screening and application of Nocardia markers. BACKGROUND

[0002] Nocardia is a gram-positive bacteria with branched mycelium. Clinically, Nocardia infection is relatively rare, usually involving acute or chronic pulmonary infection. Nocardia widely exists in soil, rotten vegetation, animal manure, fresh water and seawater and the like. It was first isolated from a cow by Nocard in 1888. It is mainly transmitted through the respiratory tract to cause lung abscess in humans and animals, and can also cause severe opportunistic infection of human organs (including lung, skin, central nervous system and the like) through wound or blood transmission and the like. The susceptible population mainly includes groups with low immune function (such as combined with AIDS, organ transplantation, diabetes) and occasionally normal immune function groups. Although identification of Nocardia at the species level is crucial for evaluating pathogenicity, predicting antimicrobial susceptibility and prognosis, the classification of Nocardia has been controversial. There are many different species in the genus Nocardia, and some common pathogenic Nocardia species in clinic include Nocardia farcinica, Nocardia gilversonkii, Nocardia brasiliensis, Nocardia auris and the like. Different Nocardia species can exhibit different pathogenicity and pathological characteristics. Nocardia farcinica and Nocardia gilversonkii are the most common species causing pulmonary infection, and Nocardia brasiliensis mainly causes skin and soft tissue nocardiosis. And strains in different regions and environments have different virulence and infection characteristics.

[0003] Nocardiosis has similarities with common bacterial and fungal infections, and the clinical manifestations mainly include non-specific infection symptoms such as cough, fever and chest pain. Using traditional methods for diagnosis, smear shows weak acid-fastness and is easy to be confused with Mycobacterium tuberculosis, so the identification of these pathogenic bacteria is often confusing, which is easy to cause misdiagnosis, so as to delay the best treatment opportunity and lead to high mortality. Molecular diagnosis methods include gene sequencing, high-throughput sequencing technology, multi-locus sequence analysis and the like. Although the molecular diagnosis method has high accuracy, the cost is high and the accessibility is low. At the same time, different Nocardia species have different antimicrobial susceptibility patterns, and identification of Nocardia at the species level is crucial for evaluating the sensitivity of antimicrobial drugs and pathogenicity. However, the genomic sequences of Nocardia species are highly similar, and biochemical identification methods are insufficient to distinguish the species. 16S rDNA sequencing is a commonly used method with high accuracy, but the time consumption and equipment complexity limit its application in conventional laboratories. Therefore, it is necessary to study a simple, rapid and accurate marker for identification and detection of Nocardia, so as to early diagnose, timely and correctly treat diseases caused by Nocardia, improve the cure rate and improve the prognosis.

[0004] To meet the urgent need for rapid identification of Nocardia, several emerging technologies and methods have been applied in recent years to improve accuracy and efficiency in the identification and diagnosis of Nocardia. Advances in genomics technology have provided new possibilities for the rapid identification of Nocardia. Whole-genome sequencing technology can help rapidly obtain the genome sequence of Nocardia, which can then be identified by comparing it with known Nocardia genome databases. This method can not only accurately identify the species and strains of Nocardia, but also help analyze their drug resistance and pathogenic mechanisms, providing more precise guidance for clinical treatment. Summary of the Invention

[0005] In a first aspect, the present invention provides a biomarker for identifying and detecting Nocardia, the biomarker being F6W96_34950, which may be a nucleic acid sequence or an amino acid sequence.

[0006] Furthermore, the nucleic acid sequence of the biomarker F6W96_34950 is shown in SEQ ID NO.1.

[0007] Furthermore, the amino acid sequence of the biomarker F6W96_34950 is shown in SEQ ID NO.2.

[0008] Furthermore, the Nocardia species include, but are not limited to, Nocardia asteroides, Nocardia short chain, Nocardia glanders, Nocardia flesh-colored, Nocardia brasiliensis, Nocardia blueberry, Nocardia guinea pig otitis media, Nocardia africana, Nocardia bitter, Nocardia gelsenkirchen, and Nocardia dermatophytes.

[0009] Secondly, the present invention provides primers for identifying and detecting a biomarker of Nocardia, wherein the biomarker is F6W96_34950, and the primer sequence of the biomarker is shown in SEQ ID NO.3-4.

[0010] 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 F6W96_34950 and the biomarker may be a nucleic acid sequence or an amino acid sequence.

[0011] Furthermore, the nucleic acid sequence of the biomarker F6W96_34950 is shown in SEQ ID NO.1.

[0012] Furthermore, the amino acid sequence of the biomarker F6W96_34950 is shown in SEQ ID NO.2.

[0013] Furthermore, if the patient's biological sample contains the nucleic acid or amino acid sequence of the biomarker F6W96_34950, it is determined that the patient's biological sample contains Nocardia.

[0014] Further, the biological sample of the patient is selected from one or more of sputum, alveolar lavage fluid, pleural effusion, cerebrospinal fluid, and blood.

[0015] In a fourth aspect, the present application provides a kit for detecting biomarkers of Nocardia, wherein the kit comprises reagents for detecting the biomarker of the first aspect, and the biomarker is the nucleic acid or amino acid sequence of F6W96_34950.

[0016] Further, the nucleic acid sequence of the biomarker F6W96_34950 is shown as SEQ ID NO. 1; and the amino acid sequence of the biomarker F6W96_34950 is shown as SEQ ID NO. 2.

[0017] Further, the detection reagent of the biomarker comprises the primer sequences shown as SEQ ID NO. 3-4.

[0018] In a fifth aspect, the present application provides a screening method for detecting biomarkers of Nocardia, wherein the method comprises the following steps:

[0019] S1. obtaining samples to obtain Nocardia strains at the genomic or chromosomal level of complete or assembled genomic sequences as a discovery group and a validation group;

[0020] S2. annotating the sample genomes;

[0021] S3. performing pan-genome analysis on the annotated genomic sequences of the discovery group in S2, and performing comparative genome analysis with the validation group to discover specific genes;

[0022] S4. designing primers based on the selected specific genes for diagnosing Nocardia, and performing sequence alignment analysis of the obtained primers in a database to finally obtain specific biomarkers.

[0023] Further, the genomic data annotation comprises core genes, non-core genes, and specific genes.

[0024] Further, the core genes refer to genes present in all animal and plant strains or strains; the non-core genes refer to genes present in 1 or more animal and plant strains or strains; and the specific genes refer to genes present only in one strain.

[0025] Further, the pan-genome analysis is to align the obtained specific genes with the annotated gene sequences of the validation group.

[0026] Further, Prokka software is selected for genome data annotation, BPGA software is selected for pan-genome analysis, and BLAT software is selected for gene sequence alignment.

[0027] Further, the gene sequence alignment is performed for gene homology analysis and comparative gene analysis with a homology threshold of 50% and a coverage of 50%.

[0028] Further, the biomarker is F6W96_34950, and the upstream and downstream primer sequences of the biomarker are shown in SEQ ID NO. 3-4.

[0029] Beneficial effects:

[0030] The exclusive molecular marker of the common pathogenic Nocardia in the present application is F6W96_34950, and by recognizing the molecular marker, the common pathogenic Nocardia can be accurately identified and detected, thereby at least partially achieving the differential diagnosis of diseases caused by the common pathogenic Nocardia, improving the cure rate and improving the prognosis. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Pan-genome gene accumulation curve and core genome gene accumulation curve diagram;

[0032] Figure 2 Primer pair verification and detection result map based on F6W96_34950. DETAILED DESCRIPTION

[0033] The specific embodiments of the present disclosure are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.

[0034] The raw materials, reagents, instruments and equipment involved in the embodiments of the present application can be obtained by purchase, unless otherwise specified.

[0035] Example 1: Screening of biomarkers of Nocardia

[0036] 106 strains of Nocardia strains with complete genome sequences at the genome or chromosome level were collected, including 33 strains of Nocardia, 31 strains of non-tuberculous Mycobacterium, 23 strains of Mycobacterium tuberculosis, 12 strains of Gordonia, and 7 strains of Tsukamurella, as a discovery group, which involves 5 species in total.

[0037] In addition, 496 strains with original sequencing reads were collected, including 297 strains of Nocardia, 108 strains of non-tuberculous Mycobacterium, 39 strains of Mycobacterium tuberculosis, 29 strains of Gordonia, and 23 strains of Tsukamurella, as a first verification group.

[0038] The genomic data of the discovery group and the verification group were quickly and efficiently functionally annotated by using the Prokka prokaryotic genome rapid annotation software; the gene sequences of the discovery group after annotation were subjected to pan-genome analysis by using the BPGA software (see Figure 1 ), the gene set clustering of each species was performed, the core genes, non-core genes and specific genes of each species were found, and the specific genes obtained were subjected to alignment with the gene sequences after annotation of the verification group by using the BLAT software, and the gene homology analysis and comparative gene analysis were performed with the same identity threshold of 50% and coverage of 50%, 18 specific genes were found, which were used as biomarker candidates for diagnosis of Nocardia.

[0039] After 18 specific genes were selected as candidate markers in the experiment, primers were designed based on the gene sequences by using the primer design software Primer Premier 5.0, and the obtained primers were subjected to sequence alignment analysis in the NCBI database (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), the specific gene sequences with high homology to other species were excluded, and the non-specific matching of the designed primers with other species sequences was also excluded, finally, one specific biomarker was obtained (the nucleic acid sequence of the biomarker is shown in Table 1, and the amino acid sequence is shown in Table 2), and the specific amplification primers after optimization are shown in Table 3.

[0040] Table 1 Nucleic acid sequence of biomarker

[0041]

[0042]

[0043] Table 2 Amino acid sequence of biomarker

[0044]

[0045] Table 3 Biomarker primer pair sequence table

[0046]

[0047] Example 2 Detection effect of biomarker on Nocardia

[0048] 24 strains of Nocardia and other strains were collected, and the genomic DNA of the strains was extracted by using the DNA extraction kit of Qiagen company according to the instructions. The concentration and purity of the genomic DNA were determined by using ultraviolet spectrophotometer. Various genomic DNAs were small amount of subpackaged and stored at -20℃ for standby.

[0049] PCR amplification reaction procedure is 94℃ 5min, 94℃ 30s, 68℃ 30s, 72℃ 30s (40 cycles), 72℃ 2min, and PCR amplification product is obtained. (The primer pair of the specific molecular marker of Nocardia includes the primer shown in SEQ ID NO. 3-4, wherein the primer pair sequence is shown in Table 3).

[0050] A 1.5% agarose gel is prepared by a conventional method, 5ul sample amplification product is added into a gel loading well, 5ul DNA marker is added into another well, and electrophoresis is carried out at 120v for about 30min. The electrophoresis result is observed under ultraviolet light. When the agarose gel detection is carried out, the strain order and result of the sample added into the sample well are shown in Table 4, and the electrophoresis result is shown in Figure 2. Figure 2

[0051] Table 4 Strain order and result of sample added into the sample well of 24 strains of Nocardia and other strains

[0052]

[0053]

[0054] It can be known from Table 4 and Figure 2 The biomarker provided by the application can realize the detection of Nocardia, and the process is simpler, faster, more accurate and lower in cost.​

Claims

1. A biomarker for differentiating Nocardia, the biomarker being F6W96_34950; the nucleic acid sequence of the biomarker F6W96_34950 is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO.

2. 2.A primer of a biomarker for differentiating Nocardia, the biomarker being F6W96_34950; the nucleic acid sequence of the biomarker F6W96_34950 is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; the primer sequence of the biomarker is shown as SEQ ID NO. 3-4. 3.Use of a reagent for detecting a biomarker in the preparation of a kit for differentiating Nocardia, the biomarker being F6W96_34950; the nucleic acid sequence of the biomarker F6W96_34950 is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; the primer sequence of the biomarker is shown as SEQ ID NO. 3-4.

4. Use according to claim 3, wherein the compound is ###0002### When a patient's biological sample contains the nucleic acid or amino acid sequence of the biomarker F6W96_34950, it is determined that the patient's biological sample contains Nocardia. 5.The use of claim 3, wherein the patient's biological sample is selected from one or more of sputum, alveolar lavage fluid, pleural effusion, cerebrospinal fluid and / or blood. 6.A kit for detecting a biomarker for differentiating Nocardia, the kit containing a reagent for detecting the biomarker of claim 1, the biomarker being the nucleic acid or amino acid sequence of F6W96_34950; the nucleic acid sequence of the biomarker F6W96_34950 is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; the detection reagent of the biomarker contains the primer sequence shown as SEQ ID NO. 3-4.

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