CN113151516A - Human high-risk zoonosis type streptococcus suis specific sequence, detection primer and application - Google Patents
Human high-risk zoonosis type streptococcus suis specific sequence, detection primer and application Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a human high-risk zoonosis type streptococcus suis specific sequence and application thereof, wherein the specific sequence is shown as SEQ ID NO. 1. Designing a detection primer aiming at the sequence: TGACAAGGTATTTGGGTGGGATG and AAGAAGGTCGTAGTTCTGGGAGC, the primer has good identification effect on human-derived isolated streptococcus suis and virulent streptococcus suis, and all positive samples with strips can see color reaction under visual observation, so that the detection method established based on the specific sequence provided by the invention can be well applied to the clinical detection process.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a streptococcus suis specific sequence of a human high-risk zoonosis type, a detection primer and application.
Background
The streptococcus suis is an important zoonosis pathogen, and can cause meningitis, septicemia, arthritis and the like when infecting human beings and also can infect pigs, thereby causing serious economic loss to the pig industry. Streptococcus suis often causes sporadic infections in humans, and causes two outbreaks in china, which is a worldwide concern. The emergence of resistant strains will pose a greater threat to the health of humans and animals. Since the first case of human streptococcus suis infection reported in denmark in 1968 (Perch et al, 1968), there were 34 cases of human streptococcus suis infection found in a total of 34 countries worldwide, including europe, asia, north america, south america, australia, new zealand, and so on, with asia being the most severe (Goyette-Desjardins et al, 2014) (Ye et al, 2008) (Takeuchi et al, 2012); mai et al collected more than 450 cases of bacterial meningitis in the southern vietnam, with up to 151 cases due to streptococcus suis, the leading cause of adult bacterial meningitis in this area (Mai et al, 2008); ma et al systematically collected and analyzed cases of Streptococcus suis infection from 1 month to 7 months 2005 in hong Kong official hospital, and found meningitis in 48% of cases, septicemia in 38% of cases, endocarditis in 14% of cases, and lethality in 5% of cases (Ma et al, 2008). The swine streptococcosis is not only an important pathogen which harms the swine industry all over the world, but also seriously threatens the physical health and life safety of human beings.
Studies have shown that almost 100% of pig farms detect positivity to streptococcus suis (Segura et al, 2017), which is mainly distributed in the upper respiratory tract sites of pigs. The high positive rate of Streptococcus suis in pig farms (Segura et al 2017; Goyette-Desjardins et al 2014; Gottschalk et al 2010) tends to lead to high carriage rates of pork products and mutual contamination between foods, thereby increasing the risk of infection of humans with Streptococcus suis. Therefore, it is important to develop a detection technology for distinguishing the zoonotic streptococcus suis from the nonpathogenic streptococcus suis.
In the 80-90 s, a total of 35 serotypes (including types 1-34, 1/2) were identified in Streptococcus suis (Okura et al, 2016). Recent studies have shown that serotypes 20, 22, 26, 32, 33 and 34 do not belong to Streptococcus suis (Tien le et al, 2013; Hill et al, 2005). Although serotypes 2 and 14 are considered to be the major serotypes of infected humans, no current study has shown that serotypes have a very high correlation with whether they are capable of infecting humans (Goyette-Desjardins et al, 2014). For example, there are 5 cases of human infection caused by serotypes, serotype 4 (Arends & Zanen, 1988), serotype 5 (Kerdsin et al, 2011), serotype 16 (Hampson et al, 1993), type 21 (Callejo et al, 2014) and type 24 (Kerdsin et al, 2011), respectively; and since 2011, cases of human streptococcus suis infection have been reported for the first time in cambodia, chile, francis, and korea (Goye tte-Desjardins et al, 2014).
The whole genome sequencing and typing technology can perform epidemiological analysis on the basis of the whole genome base sequence and comprehensively reflect the heredity and variation characteristics of pathogenic bacteria. Compared with the traditional molecular typing methods such as Pulse Field Gel Electrophoresis (PFGE), bacterial multi-locus sequence typing (MLST), multi-locus variable number tandem repeat sequence analysis (MLVA) and the like, the whole genome sequencing typing technology improves the resolution of strain evolution and traceability analysis and is beneficial to rapidly confirming and tracking pathogens. In recent years, the technology is widely applied to researches on epidemiology, pathogenicity, evolution and the like of streptococcus suis. Weinert et al performed whole genome sequencing of 191 Streptococcus suis from Vietnam and analyzed the genetic differences between the human and porcine strains, and did not find key factors for host specificity (Weine rt, et al 2015). Although the subsequent analysis of this study designed molecular targets for invasive pathogenic streptococcus suis and only performed clinical tests for porcine-derived strains, it cannot be said whether the molecular targets could be applied to the detection of high-risk zoonotic strains in humans (wileman et al, 2019). Willemse et al also performed genome sequencing and analysis of 98 S.suis strains in the Netherlands, of which 24 strains were isolated from a human source, and although some related genetic islands and prophages were also found that were specifically present in some of the zoonotic group strains, these strain genetic factors were apparently not suitable as detection targets for the entire zoonotic strain (Willemse, et al.2016), and the source of the studied strain had great geographical limitations and was not suitable for identifying molecular detection targets for the prevalent zoonotic strain.
Disclosure of Invention
The invention aims to provide a human high-risk zoonosis type streptococcus suis specific sequence, which is shown as SEQ ID NO. 1.
The invention also aims to provide application of a reagent for detecting the gene shown in SEQ ID NO.1 in preparation of a detection kit for human high-risk zoonosis type streptococcus suis.
In order to achieve the purpose, the invention adopts the following technical measures:
screening of human and livestock high-risk zoonotic streptococcus suis specific sequences, the applicant analyzes the currently disclosed streptococcus suis and a whole genome sequence of the streptococcus suis collected by the applicant, and constructs a phylogenetic tree based on a whole-gene multi-site sequence typing method, the typing method divides the 1634 strains into three types, screens specific genes in 3 Clades respectively, screens 35 group-specific genes in total, wherein 25 in CladeA and 10 in CladeB1 have no group-specific genes found in CladeB2, and screens out the specific sequences shown in SEQ ID No.1 from CladeA through final PCR effect screening.
The application of the reagent for detecting the gene shown in SEQ ID NO.1 in preparing the detection kit for the human high-risk zoonosis streptococcus suis comprises the steps of detecting whether the DNA of a target streptococcus suis contains the gene shown in SEQ ID NO.1 or not in a conventional mode in the field, so that whether the strain belongs to the human high-risk zoonosis streptococcus suis or not can be judged;
in the above application, preferably, the reagent is a primer, and the primer is: TGACAAGGTATTTGGGTGGGATG, and AAGAAGGTCGTAGTTCTGGGAGC.
Compared with the prior art, the invention has the following advantages:
the invention discloses a target sequence related to zoonosis type streptococcus suis for the first time, and the sequence is used as a target by utilizing a conventional scheme in the field, so that the zoonosis type streptococcus suis can be identified with high probability, and the application prospect is great.
Drawings
Figure 1 shows the topology of an unrooted tree showing the population structure of 3 different streptococcus suis.
FIG. 2 shows the PCR amplification using representative strains from different Clade to verify the reliability of the molecular targets.
FIG. 3 is a diagram showing the result of amplification of a test strain using a specific sequence primer.
Detailed Description
The technical scheme of the invention is a conventional scheme in the field if not particularly specified; the reagents or materials, if not specifically mentioned, are commercially available.
Example 1:
obtaining a specific sequence of the streptococcus suis with high-risk zoonosis type for human:
the sequence alignment samples in this example are:
the strains used for genome analysis in the present application included 1634 strains, 144 of which were Streptococcus suis strains self-collected and sequenced by the applicant, including Streptococcus suis isolates 103 strain and sick swine isolates 41 strain; 1490 published genomic data of Streptococcus suis were downloaded from NCBI database. All strains use the streptococcus suis BM407 as a reference genome to carry out multi-sequence alignment, and generate a multi-sequence alignment file for phylogenetic analysis. Compared with the traditional molecular typing method, the phylogenetic tree constructed by the method based on the whole-gene multi-site sequence typing has higher resolution. The evolutionary tree constructed by the analysis has 3 obvious branches (figure 1), wherein one branch comprises almost all human strains (96 percent, 549/570) which are defined as a human high-risk group Clade A, the other two branches are Clade B1 and Clade B2 respectively, 69 percent (181/262) of the strains in the Clade B1 are sick pig isolates which are defined as sick pig related groups, 279 strains (72.2 percent) of all 386 healthy pig isolates are clustered in Clade B2, the proportion of the healthy pigs reaches 79 percent, and the healthy pig related groups are defined.
Applicants further screened the differential genes, defining the grouping-specific genes as: the target genes exist in more than 95 percent (including) of strains in the group, the proportion of the target genes in other groups is less than 5 percent (including), 35 group-specific genes are screened according to the screening of the genes which are respectively unique in 3 Clades, 25 in CladeA and 10 in CladeB1 are obtained, the group-specific genes are not found in CladeB2, 25 candidate sequences are screened from CladeA finally through the final PCR effect screening, and finally 2 specific sequences are selected, wherein one of the specific sequences is shown in SEQ ID NO. 1.
Example 2:
the application of the specific sequence in identifying the zoonosis type streptococcus suis comprises the following steps:
this example verifies the accuracy of the specific sequence selected in example 1 by PCR
Primers designed against the specific sequences obtained in example 1 were as follows:
PCR conditions were as follows: initial PCR was performed using a 25. mu.L system with 2. mu.L of DNA template, 1. mu.L of each of the upstream and downstream primers, Mix 12.5. mu.L, dd H2O8.5 mu L; the reaction conditions are as follows: 5min at 95 ℃; 30s at 95 ℃, 30s at 55 ℃ and 2min at 72 ℃ for 35 cycles; finally, extension was carried out at 72 ℃ for 7 min. The PCR product was sent to Biotech for sequencing.
The sample to be tested is:
validation of the accuracy of the genome prediction was performed using 24 strains derived from 3 Clades:
these 24 representative strains were: the first group of strains: 6 healthy pig isolates clustered in Clade A (1024-1, 1358, 2283, 2355, 812, 832); a second group of strains: 2 sick pig isolates and 4 humanized isolates clustered in Clade A (SC19, P1/7, CH52, CH61, CH53, LSM 102); a third group of strains: 6 healthy pig isolates clustered in Clade B2 (1247, 13, 1783, 1053, 3096, 1100); fourth group of strains: 6 sick pig isolates clustered in Clade B1 (CPD1, CPD22, CPD23, CPD24, CPD26, CPD 33).
As shown in FIG. 2, the G20 Primer derived from CladeA was able to detect well 12 strains of the group (including 6 healthy pig strains); at the same time, strains from Clade B1 and Clade B2 could not be detected.
Example 3:
the clinical application of the specific sequence in identifying the zoonosis type streptococcus suis is as follows:
the test samples in this example were as follows, and the PCR conditions and primers were the same as in example 2:
the sample for detecting the streptococcus suis in the present case comprises 43 strains, wherein 21 human streptococcus suis are clinical strains newly separated in a laboratory. In addition 22 strains were published by Nanjing university of agriculture and were virulence verified using animal models, including 12 strains of virulent strain (1 isolate of human origin, 9 isolates of sick pigs, 2 isolates of healthy pigs and 10 strains of avirulent strain (3 isolates of sick pigs, 7 isolates of healthy pigs) (Qian et al, 2018; Yu et al, 2016).
The above strains were subjected to DNA extraction and PCR amplification using G20 Primer according to the method in example 2. .
The results showed that all of the primers were able to identify 21 isolates of human origin (A in FIG. 3); at the same time, 12 virulent strains (A in FIG. 3) could be detected, but 10 less virulent strains (B in FIG. 3) could not be detected. Therefore, the primer has good identification effect on the human-derived isolate strain and the virulent strain. All positive samples with strips can see color reaction under the visual observation, so that the method can be well applied to the clinical detection process.
Sequence listing
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Claims (3)
1. An isolated high-risk zoonotic streptococcus suis specific gene of human, which is shown as SEQ ID NO. 1.
2. The application of a reagent for detecting the gene shown in SEQ ID NO.1 in preparing a detection kit of human high-risk zoonosis type streptococcus suis.
3. The use of claim 2, wherein the reagent is a primer comprising: TGACAAGGTATTTGGGTGGGATG, and AAGAAGGTCGTAGTTCTGGGAGC.
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| 马青;刘英;王月;李世军;唐光鹏;王定明;: "贵州省死亡病例1型猪链球菌的分离及分子生物学特征分析", 现代预防医学, no. 03 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151309A (en) * | 2021-04-01 | 2021-07-23 | 华中农业大学 | Human high-risk zoonosis type streptococcus suis specific sequence and application |
| CN113151309B (en) * | 2021-04-01 | 2023-10-03 | 华中农业大学 | Streptococcus suis specific sequence with high risk of human beings and livestock and application thereof |
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