High-throughput qPCR detection primer-probe system and detection method for rimerella anatipefae

Abstract

The application discloses a duck Riemerella anatipestifer high-throughput qPCR detection primer-probe system and a detection method. The highly conservative region is selected and designed through bioinformatics analysis of the whole genome sequence of the duck Riemerella anatipestifer. The primer-probe system comprises a primer pair and a probe for detecting the duck Riemerella anatipestifer. The primer pair is a forward primer shown in SEQ ID NO: 1 and a reverse primer shown in SEQ ID NO: 2. The probe is a probe shown in SEQ ID NO: 3. The application can realize full coverage of 216 strains of duck Riemerella anatipestifer, greatly reduce the missed detection rate, reach 100% specificity, effectively prevent cross reaction, accurately detect early infection and latent carrier condition, and has better detection performance than most traditional PCR methods. The whole qPCR detection process can be completed within 2 hours, and the operation is simple, so that standardized operation and rapid promotion can be realized in detection laboratories at all levels.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology for animal pathogenic microorganisms, and in particular to a primer-probe system and detection method for high-throughput qPCR detection of Riemerella anatipestifer. Background Technology

[0002] Riemerella anatipestifer is a significant pathogenic bacterium threatening waterfowl farming, and the core pathogen causing infectious serositis in ducks, geese, and other waterfowl. This pathogen easily spreads among farmed waterfowl, causing typical symptoms such as serositis, arthritis, and pericarditis. Not only is the morbidity rate consistently high, but the mortality rate is also substantial. An outbreak in a farm can lead to mass deaths of susceptible ducklings and goslings, as well as stunted growth and reduced quality in surviving waterfowl, resulting in significant economic losses for large-scale, intensive waterfowl farming and becoming a major bottleneck restricting the healthy development of the industry. Therefore, rapid, accurate, and efficient pathogen detection and early diagnosis of Riemerella anatipestifer are crucial for timely implementation of control measures such as isolation, disinfection, and medication to block pathogen transmission, reduce epidemic losses, and ensure the safety of aquaculture production.

[0003] Real-time quantitative PCR (qPCR) technology, with its advantages of fast detection speed, high sensitivity, strong specificity, and quantifiable results, has been widely used in the pathogen detection of *R. anatidae*, becoming a common technique for this bacterium. However, currently available qPCR detection methods for *R. anatidae* have significant limitations in primer design. Most methods design primers based on a single conserved gene sequence of the bacterium, and some optimized methods only select a small number of strains to verify primer specificity and effectiveness, failing to fully consider the species characteristics of *R. anatidae*: *R. anatidae* has a rich diversity of serotypes. More than 21 serotypes have been identified, and there is significant genetic diversity among different serotypes, with certain differences in gene sequences. In addition, variant strains of the bacterium exist in the breeding environment, making it difficult for existing detection methods to comprehensively cover *R. anatidae* strains with different serotypes and genetic backgrounds. In actual farm testing applications, these methods are prone to missing detections and producing negative results for some serotypes or variant strains, posing a high risk of missed detection. They cannot meet the actual needs of farms for comprehensive and accurate detection of the pathogen, nor can they provide reliable testing data support for epidemic prevention and control.

[0004] Based on this, and considering the serotype distribution characteristics and genetic diversity of Riemerella anatipestifer, the development of a qPCR detection scheme that has undergone systematic validation by isolating strains of different serotypes, regions, and breeding environments on a large scale, and that has extremely high detection coverage and no missed detection for various strains of this bacterium, has become an urgent need in the field of disease prevention and control in waterfowl farming. It has important practical significance and application value for improving the detection accuracy of Riemerella anatipestifer, perfecting the comprehensive prevention and control system for infectious serositis in waterfowl, and promoting the high-quality development of the waterfowl farming industry. Summary of the Invention

[0005] The purpose of this invention is to provide a high-throughput qPCR detection primer-probe system and detection method based on *Riemerella anatipestifer*, and to establish a standardized procedure for inclusive validation using 352 clinical isolates covering different serotypes and geographical origins, ensuring the broad-spectrum, reliable, and practical nature of the detection method. This application can achieve full coverage of 216 *Riemerella anatipestifer* strains, significantly reducing the false negative rate; it achieves 100% specificity, effectively eliminating cross-reactivity; it can accurately detect early infection and latent carrier status, and its detection performance is superior to most traditional PCR methods; the entire qPCR detection process can be completed within 2 hours, is simple to operate, and facilitates standardized operation and rapid promotion by testing laboratories at all levels.

[0006] This invention discloses a primer-probe system for high-throughput qPCR detection of Riemerella anatipestifer. The system utilizes bioinformatics analysis of the whole genome sequence of Riemerella anatipestifer to screen and select highly conserved regions for design. The primer-probe system includes primer pairs and probes for the specific detection of Riemerella anatipestifer. The primer pairs are the forward primer shown in SEQ ID NO:1 and the reverse primer shown in SEQ ID NO:2, and the probe is the probe shown in SEQ ID NO:3.

[0007] Specifically, the forward primer is SEQ ID NO:1:5′-ATATTTACCTACCGGCTTC-3′; Reverse primer SEQ ID NO:2:5′-AACAGGAATWAGTGCCGA-3′; W = A / T degeneracy; Probe SEQ ID NO:3:5′-GAARGTAGCCACTTCGTACAC-3′; R=A / G degeneracy.

[0008] Preferably, the 5′ end of both the primer pair and the probe is modified with a fluorescent group, and the 3′ end is modified with a quenching group.

[0009] In any of the above schemes, a preferred embodiment is to perform multiple sequence alignment on the gene sequences of multiple strains of *Riemerella anatipestifer*, and select a conserved sequence of 165 bp located at 767058-767223 bp in the full sequence as the target fragment. The sequence of the target fragment is shown in SEQ ID NO:4. To target the relatively stable mutations of the bases in the target fragment in the *Riemerella anatipestifer* population, degenerate bases are introduced at the corresponding positions of the primer and probe sequences. The A / T mutation site at the 9th position of the 3' end of the downstream primer is designed as "W", and the A / G mutation site at the 4th position of the 5' end of the probe is designed as "R".

[0010] The present invention also discloses a high-throughput qPCR detection kit based on Riemerella anatipestifer, including the primer-probe system described above.

[0011] The present invention also discloses the use of the above-mentioned primer-probe system or kit in the detection of Riemerella anatipestifer for non-diagnostic and non-therapeutic purposes.

[0012] The present invention also discloses a method for detecting Riemerella anatipestifer for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: using the primer-probe system or kit described above to perform a nucleic acid amplification reaction on the sample to be tested, and determining whether the sample to be tested contains Riemerella anatipestifer based on the amplification results.

[0013] This invention also discloses a method for high-throughput qPCR detection of Riemerella anatipestifer using the above primer-probe system, comprising the following steps: (1) Template preparation and strain library construction: Collect the gene sequences of Riemerella anatipestifer from the gene library, as well as the identified clinical isolates and closely related non-target bacteria of Riemerella anatipestifer, and extract the genomic DNA of each strain to construct a strain genomic template library; (2) Detection by probe-based qPCR: The template was detected by probe-based qPCR. The total volume of the reaction system was 20 μL, which contained 2 × All-Powerful qPCR PreMix, primer-probe mixture and template DNA. (3) Amplification reaction conditions: incubation at 37℃ for 2 min; pre-denaturation at 95℃ for 30 s; followed by 45 amplification cycles, each cycle including denaturation at 95℃ for 10 s and annealing extension at 52.7℃ for 30 s.

[0014] Preferably, after amplification detection, result determination and data analysis are performed, specifically including: Positive result determination: When the Ct value is ≤35 and the amplification curve shows a typical logarithmic growth phase, it is considered positive; Inclusivity calculation: Calculated according to the formula "Inclusivity (%) = Number of positive strains / Total number of target strains × 100%"; Specificity assessment: Observe the amplification of all closely related non-target strains to determine whether non-specific amplification occurs.

[0015] In any of the above schemes, the closely related non-target bacteria in step (1) include one or more of Escherichia coli, Salmonella, Streptococcus, and Acinetobacter. Beneficial effects

[0016] This invention discloses a high-throughput qPCR detection primer-probe system and validation method based on *Rimerella anatipestifer*. Through bioinformatics analysis of the whole genome sequence of *Rimerella anatipestifer*, highly conserved regions were selected for design. The primer-probe system includes primer pairs and probes for detecting *Rimerella anatipestifer*. The primer pairs are the forward primer shown in SEQ ID NO:1 and the reverse primer shown in SEQ ID NO:2, and the probe is the probe shown in SEQ ID NO:3. Specific effects are as follows: (1) High coverage: This application can achieve full coverage of 216 strains of Riemerella anatipestifer, and can detect almost all known serotypes and genetically variant strains, greatly reducing the false negative rate, and is especially suitable for epidemiological surveys and identification of unknown strains; (2) Excellent specificity and sensitivity: 100% specificity, which can effectively eliminate cross-reaction; single-copy sensitivity, which can accurately detect early infection and latent bacterial carriers, and its detection performance is better than most traditional PCR methods. (3) Fast, efficient and easy to standardize: The entire qPCR detection process can be completed within 2 hours. With the use of validated ready-to-use primer-probe dry powder, the operation is simple and convenient for testing laboratories at all levels to achieve standardized operation and rapid promotion. (4) Significant application value: It can be directly used for the development of diagnostic reagent kits and can be widely used in scenarios such as rapid on-site diagnosis in farms, quarantine of related products, and evaluation of vaccine immunization effects, with both good economic and social benefits. Attached Figure Description

[0017] Figure 1 This is a partial coverage result of the detection method of the present invention used to detect 216 strains of Riemerella anatipestifer. Figure 2 This is another part of the coverage results for detecting 216 strains of Riemerella anatipestifer using the detection method of the present invention; Figure 3 This is a further partial coverage result of the detection method of the present invention used to detect 216 strains of Riemerella anatipestifer; Figure 4 This is a distribution diagram showing the specificity of the detection method of the present invention for detecting 216 strains of Riemerella anatipestifer. Figure 5 This is a set of standard curve results for qPCR detection in this invention. The graph is plotted as the logarithm of the standard concentration (log 10 The copy number is used as the X-axis, and Ct is used as the Y-axis. Figure 6 This is another set of standard curve results for the qPCR detection of this invention. This graph uses... Figure 5 The Ct values ​​of each concentration point on the standard curve were plotted based on a single detection. Figure 7 The graph shows the sensitivity test results using standard DNA at six concentration gradients with a 10-fold serial dilution as templates. Figure 8 The graph shows the sensitivity test results using standard DNA at four concentration gradients with a 2-fold serial dilution as templates. Figure 9 The amplification curves were constructed using data analysis software, specifically using four concentration gradient standards diluted 2-fold as templates, to construct the amplification curves between Ct values ​​(X-axis) and relative fluorescence intensity increases (Y-axis). Figure 10 This is a repeatability standard curve diagram of the detection method of the present invention; Figure 11 This is a schematic diagram of the base degeneracy positions of the oligonucleotide probe of the present invention. Detailed Implementation

[0018] To better understand the technology and advantages of the present invention, the present invention will be further described below through specific embodiments and in conjunction with the accompanying drawings.

[0019] This invention discloses a high-throughput qPCR detection primer-probe system based on Riemerella anatipestifer. The system is designed by selecting highly conserved regions through bioinformatics analysis of the whole genome sequence of Riemerella anatipestifer. The primer-probe system includes primer pairs and probes for detecting Riemerella anatipestifer. The primer pairs are the forward primer shown in SEQ ID NO:1 and the reverse primer shown in SEQ ID NO:2, and the probe is the probe shown in SEQ ID NO:3.

[0020] Specifically, the forward primer is SEQ ID NO:1:5′-ATATTTACCTACCGGCTTC-3′; Reverse primer SEQ ID NO:2:5′-AACAGGAATWAGTGCCGA-3′; Probe SEQ ID NO:3:5′-GAARGTAGCCACTTCGTACAC-3′.

[0021] Primer design was based on large-scale genome analysis. Highly conserved gene sequences within the *R. anatidae* species were screened from 331 *R. anatidae* gene sequences (downloaded from NCBI) and 352 clinical isolates in the gene bank as detection targets. After multiple sequence alignment of multiple *R. anatidae* gene sequences, a conserved sequence of 165 bp located at positions 767058-767223 bp was selected as the target fragment, as shown in SEQ ID NO:4. Degenerate bases were introduced at corresponding positions in the primer sequences to target relatively stable mutations present in the population. Specifically, the target fragment contains a degenerate base at position 40. The A / T variant site at position 9 of the 3' end of the downstream primer was designed as "W" (representing A or T) to cover known single nucleotide polymorphism (SNP) sites of the target gene. This design ensures primer compatibility with all known variant types, thereby guaranteeing the stable amplification capability and inclusiveness of the detection method for all target strains.

[0022] This invention also discloses an oligonucleotide probe, the sequence of which is shown in SEQ ID NO:3, containing a sequence having at least 95% identity with the sequence shown in SEQ ID NO:4, wherein the A / G variant site located at position 4 of the 5′ end of the probe is designed as “R” (representing A or G). The sequence contains a degenerate base at position 157 to cover known single nucleotide polymorphism (SNP) sites of the target gene. Specific degenerate positions and optional bases are as follows... Figure 11 As shown in Table 1 below: Table 1 Note: IUPAC encoding is an internationally recognized degenerate base code, such as R=A / G, Y=C / T, S=G / C, W=A / T, K=G / T, M=A / C, B=C / G / T, D=A / G / T, H=A / C / T, V=A / C / G, N=A / C / G / T.

[0023] This invention also discloses a detection method based on a high-throughput qPCR primer-probe system for Riemerella anatipestifer, comprising the following steps: (1) Template preparation and strain library construction: 352 identified clinical isolates of Riemerella anatipestifer (covering major serotypes and different regional sources) and several closely related non-target bacteria (such as Escherichia coli, Salmonella, Streptococcus, Acinetobacter, etc.) were collected, and genomic DNA of each strain was extracted to construct a strain genomic template library; (2) Detection using probe-based qPCR: The above template was detected using probe-based qPCR. The total volume of the reaction system was 20 μL, and the specific composition was as follows: 10 µL of 2 × All-Powerful qPCR PreMix, 9 µL of primer-probe mixture (2 µL of primers (f and r), 0.5 µL of probe, 6.5 µL of water), and 1 µL of template DNA; The reaction conditions were set as follows: incubation at 37℃ for 2 min; followed by pre-denaturation at 95℃ for 30 s; then 45 amplification cycles were performed, with each cycle consisting of denaturation at 95℃ for 10 s and annealing extension at 52.7℃ for 30 s (fluorescence signals were collected during this stage); detection was completed after amplification. Control settings: Each reaction plate includes a positive control (a standard template of Riemerella anatipestifer DNA with a known copy number, i.e., RA DNA) and a template negative control (without template DNA). The control results are used to monitor the reaction specificity and contamination status to ensure the reliability of the test results. Result determination and data analysis: Positive criteria: A Ct value ≤ 35 and an amplification curve showing a typical logarithmic growth phase are considered positive; a Ct value > 35 or no amplification curve are considered negative. Inclusivity (coverage) calculation: The coverage ability of this method for different strains of Riemerella anatipestifer was evaluated by calculating "number of positive strains / total number of target strains" according to the formula "number of positive strains / total number of target strains"; Specificity assessment: Check whether non-specific amplification occurs in all closely related non-target strains. If no specific amplification occurs, it indicates that the method has good specificity.

[0024] The high-throughput qPCR detection method for Riemerella anatipestifer established in this invention has shown excellent performance across all indicators after performance verification. Specific experimental results are as follows: (1) Coverage results: The coverage test results are as follows Figure 1 , Figure 2 and Figure 3 As shown, among the 216 strains of Riemerella anatipestifer, all 216 strains showed specific amplification, with a coverage of up to 100%, which is significantly better than traditional target detection methods based on 16S rRNA, indicating that this method can effectively cover Riemerella anatipestifer strains and has wide applicability.

[0025] (2) Specificity results: Specificity verification results showed that no amplification signal was detected in any closely related non-target strains, achieving 100% specificity. The results are distributed as follows: Figure 4 As shown, this method can specifically identify Riemerella anatipestifer, with no cross-reactivity and good detection specificity.

[0026] (3) Standard curve results: The standard curve validation results are as follows. Data analysis software was used to determine the standard curve equation: Y = -3.48 * log(X) + 42.739, with the ordinate (Y) representing the Ct value and the abscissa (X) representing the initial template copy number. The amplification efficiency (Eff.) was 93.85%. A good linear relationship was observed between the logarithm of each standard concentration and its Ct value. The results are distributed as follows: Figure 5 and Figure 6 As shown: Among them, Figure 5 This is a set of standard curve results. The qPCR standard curve plot is plotted using the logarithm of the standard concentration (log...). 10 With copy number as the X-axis and Ct value as the Y-axis, the data points (red dots) show a good linear distribution. The slope of the fitted line is approximately -3.42, and the calculated amplification efficiency is 96.13%, close to the ideal amplification efficiency range. The R² value is 0.998 (>0.99), indicating extremely high linearity. The curve covers a linear range of 5 orders of magnitude (10^6). 3 -10 7 (copy / μL) Figure 6 The following is another set of standard curve results. This figure was plotted using the Ct values ​​of each concentration point in the above standard curve. The slope of the fitted straight line is approximately -3.48, the amplification efficiency is 93.85%, which is close to the ideal value, and the R² value is 1.000 (≥0.999). The above parameters indicate that the qPCR system has been well optimized.

[0027] (4) Sensitivity test results: Sensitivity verification results are as follows Figure 7 , Figure 8 As shown, six concentration gradients (2.76 × 10⁻⁶) were obtained by dilution 10-fold. 2 -2.76×10 7 (Copy / μL) standard and four concentration gradients of 2-fold serial dilution (8.9 × 10⁻⁶). 0 -7.12×10 1 Using DNA (copies / μL) as a template, an optimized quantitative real-time PCR method was employed for detection. The instrument's built-in data analysis software was used to analyze the data, establishing an amplification curve between the Ct value (X) and the increase in relative fluorescence intensity, as shown in the figure. Figure 9 As shown.

[0028] Experimental results show that the detection limit of this method can reach 8.90 copies / μL, and is within 10... 0 - 10 8 Excellent linearity within the copy / μL range (R²>0.993), such as Figure 10 As shown, it is suitable for accurate quantification of samples with extremely low loading.

[0029] Repeatability test results Table 2 Results of repeatability experiments As shown in Table 2 above, the concentration is 10 7 10 6 10 5 10 4 10 3 The gradient standard DNA at concentrations of [copy / μL] was subjected to repeatability testing by real-time PCR, and the coefficients of variation (CV) of the Ct values ​​were 0.64%, 0.66%, 0.61%, 0.36%, and 1.00%, respectively. The CV values ​​for each concentration were all less than 2%, meeting the repeatability requirements of the real-time PCR method. In summary, the real-time PCR method established in this invention has good repeatability.

[0030] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer-probe system for high-throughput qPCR detection of Riemerella anatipestifer, characterized in that: Bioinformatics analysis of the whole genome sequence of Riemerella anatipestifer was performed to screen and select highly conserved regions for design. The primer-probe system includes primer pairs and probes for specific detection of Riemerella anatipestifer. The primer pairs are the forward primer shown in SEQ ID NO:1 and the reverse primer shown in SEQ ID NO:

2. The probe is the probe shown in SEQ ID NO:

3.

2. The high-throughput qPCR detection primer-probe system based on Riemerella anatipestifer according to claim 1, characterized in that: Both the primer pair and the probe are modified with fluorescent groups at their 5' ends and quenching groups at their 3' ends.

3. The high-throughput qPCR detection primer-probe system based on Riemerella anatipestifer according to claim 1, characterized in that: After performing multiple sequence alignment on the gene sequences of multiple strains of Riemerella anatipestifer, a conserved sequence of 165 bp located at 767058-767223 bp in the full sequence was selected as the target fragment, as shown in SEQ ID NO:

4. To target the stable mutations of the bases in this target fragment in the Riemerella anatipestifer population, degenerate bases were introduced at the corresponding positions in the primer and probe sequences. The A / T mutation site at position 9 at the 3' end of the downstream primer was designed as "W", and the A / G mutation site at position 4 at the 5' end of the probe was designed as "R".

4. A high-throughput qPCR detection kit based on Riemerella anatipestifer, characterized in that: Includes the primer-probe system according to any one of claims 1-3.

5. Use of the primer-probe system according to any one of claims 1-3 or the kit according to claim 4 in the detection of Riemerella anatipestifer for non-diagnostic and non-therapeutic purposes.

6. A method for detecting Riemerella anatipestifer for non-diagnostic and non-therapeutic purposes, characterized in that, The method includes the following steps: using the primer-probe system as described in any one of claims 1-3 or the kit as described in claim 4, performing a nucleic acid amplification reaction on the sample to be tested, and determining whether the sample to be tested contains Riemerella anatipestifer based on the amplification results.

7. The primer-probe system according to any one of claims 1-3, applied to the high-throughput qPCR detection method of Riemerella anatipestifer, is characterized in that... Includes the following steps: (1) Template preparation and strain library construction: Collect the gene sequences of Riemerella anatipestifer from the gene library, as well as the identified clinical isolates and closely related non-target bacteria of Riemerella anatipestifer, and extract the genomic DNA of each strain to construct a strain genomic template library; (2) Detection by probe-based qPCR: The template was detected by probe-based qPCR. The total volume of the reaction system was 20 μL, which contained 2 × All-Powerful qPCR PreMix, primer-probe mixture and template DNA. (3) Amplification reaction conditions: incubation at 37℃ for 2 min; pre-denaturation at 95℃ for 30 s; followed by 45 amplification cycles, each cycle including denaturation at 95℃ for 10 s and annealing extension at 52.7℃ for 30 s.

8. The high-throughput qPCR detection method for Riemerella anatipestifer according to claim 7, characterized in that, After amplification detection is completed, result interpretation and data analysis are performed, including: Positive result determination: When the Ct value is ≤35 and the amplification curve shows a typical logarithmic growth phase, it is considered positive; Inclusivity calculation: Calculated according to the formula "Inclusivity (%) = Number of positive strains / Total number of target strains × 100%"; Specificity assessment: Observe the amplification of all closely related non-target strains to determine whether non-specific amplification occurs.

9. The high-throughput qPCR detection method for Riemerella anatipestifer according to claim 7, characterized in that, The closely related non-target bacteria in step (1) include one or more of Escherichia coli, Salmonella, Streptococcus, and Acinetobacter.

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