I guarantee that the research: “Evaluating the ability to detect Mycoplasma hyopneumoniae in swine of the TopSPEC® Mycoplasma hyopneumoniae qPCR KIT”was conducted by myself and that the
Equipment Sa
This article highlights essential laboratory equipment, including a centrifuge, vortex machine, level 2 biological safety cabinet, and a 20°C negative cabinet It also discusses the importance of temperature incubators and micropipettes, featuring various types such as 10 µl, 100 µl, and 1000 µl Additionally, it mentions different tips compatible with these micropipettes, along with Eppendorf tubes (1.5 ml) and the LineGene K Plus Real-Time PCR machine from Bioer, China.
In this topic, the research process is conducted according to the process in Figure 3.1.
Checking the primer specificity by bioinformatics software.
Processing and extraction of collected samples ô+
Perform real — time PCR reaction ô+
Determine the detection limit of the test kit ô
Check the analytical specificity of the test kit ô+
Evaluate the ability of the kit to detect MZ hyopneumoniae on field samples
Determine the specificity, diagnostic sensitivity, positive predictive value, negative predictive value of the test kit
Figure 3.1 The workflow of the research.
3.4.1 Checking the primer specificity by bioinformatics software
Primer-BLAST A tool for finding specific primers
Finding primers specific to your PCR template (using Primer3 and BLAST).
Retrieve recent results Publication Tips for finds tic primers (Gave search parameters) (Reset page )
PCR Template ng spec (eeetesee )
Enter accession, gi, or FASTA sequence (A :c/se‹ íc 3 preterred) @ ca) Range @ Coe)
Or, upload FASTA file Choose File | No file chosen
Use my own forward primer © CĐ
Use my own reverse primer (Š- eG
Figure 3.2 Primer-BLAST tool (Ye ef al., 2012)
The ABT company has developed primers and probes that are specifically compatible with the P97 gene of M hyopneumoniae, which has been cloned into the plasmid pUC57 To ensure the effectiveness of these primers and probes, their compatibility was assessed using Primer-Blast analysis from NCBI, confirming that the primer sequences uniquely target M hyopneumoniae and do not cause non-specific amplification with other species.
Primer-Blast not only evaluates specificity but also analyzes the melting temperature (Tm) of primers and probes to ensure they are compatible with PCR cycle conditions Additionally, it helps predict the expected size of the amplicon produced through successful amplification with verified primers and probes (Lin et al., 2023).
To process lung tissue samples using the AccuRive pDNA Prep Kit, begin by crushing 5 grams of the tissue required for testing Next, add PBS solution, mix thoroughly, and centrifuge at 14,000 rpm for 5 minutes to obtain the supernatant.
Proceed with DNA extraction according to the AccuRive pDNA Prep Kit isolation procedure The process includes steps:
Step 1: Take 200 ul DNA sample + 900 pl KTS1, take the mixture to votex and let stand for 10 minutes at room temperature.
Step 2: Add 200 ul KTS2 to the mixture then centrifuge at 13.000 rpm for 10 minutes.
To proceed, transfer 600 µl of supernatant from the centrifuged mixture into a new 1.5 ml Eppendorf tube containing 600 µl of KTS3 Gently invert the tube and allow it to stand at room temperature for 10 minutes Following this incubation, centrifuge the tube at 13,000 rpm.
10 minutes Discard the supernatant carefully so as not to distub the remaining pellet.
Step 4: Add 900 ul KTS4, continue centrifuging at 13.000 rpm for 10 minutes. Discard the supernatant carefully so as not to distub the remaining pellet Dry the tube for 10 minutes at 60°C.
Step 5: Add 50 ul KTSS to resuspend the DNA and store the DNA/RNA in -20°C refrigerator to use if not needed for the following reactions.
Real-time PCR reactions on the trial kits were performed according to the manufacturer's protocol, with the same sample volume of 5 HÌ, on the same Real-time PCR instrumentation.
The trial kit for real-time RCR reactions utilizes Taqman probes and analyzes results through two channels: FAM and HEX The FAM channel detects the pathogen M hyopneumoniae, while the HEX channel monitors the internal control The qPCR reaction is conducted in a 25 µl volume, comprising 20 µl of the M hyopneumoniae qPCR mix and 5 µl of DNA template, along with positive and negative controls.
To conduct a successful real-time PCR reaction, follow the manufacturer's recommended thermal conditions Begin with a pre-denaturation step at 95°C for 3 minutes, followed by 45 cycles alternating between 95°C for 15 seconds and 60°C for optimal amplification.
Real-time PCR results are analyzed using two color channels: FAM, which detects the amplified signal for the target DNA sample, and HEX, which captures the signal from the internal control sample A sample is considered positive for M hyopneumoniae if the FAM channel shows an amplification signal with a threshold cycle value (Ct) of 37 or less, regardless of the HEX channel's signal Conversely, a sample is deemed negative if the FAM channel fails to register a Ct value while the HEX channel does If neither channel records a Ct value, the PCR reaction is deemed failed and must be repeated.
Positive Negative Target Internal Interpretation control control control
The trial Ct value < 37 or si
(+) (-) (-) (+) Negative result Note: Ct: Threshold cycle; (+): Positive; (-): Negative
3.4.6 Determine the limit of detection (LOD)
The limit of detection (LOD) is defined as an assay's ability to identify minute concentrations of a specific substance in biological samples, and it is often equated with analytical sensitivity LOD is a crucial parameter for both qualitative and quantitative test methods, where lower detectable concentrations indicate higher analytical sensitivity In this study, the LOD was established through a real-time reaction using a trial kit and a standard solution containing the plasmid with the target gene.
M hyopneumoniae with the concentration of 10° Each reaction was performed in triplicate.
In addition, based on the processing of the reaction results of ten-fold serial plasmid dilutions with concentrations from 10° to 10° copies/ul using the charts tool of
In Microsoft Excel 365, the standard curve for the reaction was established, yielding key metrics such as the correlation coefficient (R’), the slope, and the y-intercept of the curve Additionally, the efficiency value (E) of a PCR reaction can be calculated using the formula outlined by Bustin et al (2009).
E _ : the efficiency of a PCR reaction (%)
Slope: Value of the slope of the standard curve
Analytical specificity is a crucial factor alongside analytical sensitivity in both research and practical applications It refers to an assay's ability to accurately detect the intended target without interference from similar nucleic acids or conditions related to the specimen, ensuring reliable quantification (Burd, 2010).
To assess the specificity of the test kit, conduct a Real-time PCR reaction using negative controls that encompass eight bacterial strains and three common viral strains known to cause diseases in pigs, specifically Erysipelothrix rhusiopathiae and Bordetella.
The article discusses various pathogens affecting swine, including Bordetella bronchiseptica, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis, Actinobacillus pleuropneumoniae (APP), Escherichia coli, Salmonella spp., Porcine Circovirus type 2 (PCV 2), African swine fever virus (ASF), and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) It also mentions the use of a negative control and the positive control from the testing kit.
3.4.8 Evaluation of the diagnostic performance of the trial kit for the detection of
3.4.8.1 Evaluation of the accuracy of the trial kit for the detection of
DNA extraction was conducted on six collected lung samples, followed by a Real-time PCR reaction using a test kit The results were documented, noting both negative and positive outcomes, along with the Ct values of the positive samples, which were repeated three times for accuracy.
3.4.8.2 Assessment of the stability of the trial kit for the detection of
To assess the stability of the commercial kit across multiple repetitions, the project calculated the coefficient of variation (CV%) for the Ct values of positive samples over three repetitions Additionally, the CV% of the control kit's Ct values was compared to the average Ct values obtained from the test kit.
3.4.9 Specificity, diagnostic sensitivity, positive predictive value, and negative predictive value of the trial kit
RESULTS AND DISCUSSION -. 2c c2cSccssersirrrrrrrrer pay] AT Results:of thestudy ssseessseseiseeestemasertidnobiiGBiueregaiyolgtilstoebtksotogsSkgpgdo2eSpobseertgosl 27 4.1.1 Checking the primer specificity by bioinformatics software
Determine the limit of detection (LOD) of the trial kĩt . 55555555555 28 4.1.3 Testing analytical specificity .-c0.vce.nvorsaensnerenvenointiennentatecsivsnseevenavtnenibceedsients 29 4.2 Evaluation of the diagnostic performance of the trial kit for the detection of M hyopneumoniae in field samples .c:ccecceccesceecceseeesteseeeseeseeeceeaeeeseeseenseessesees 31 4.2.1 Evaluation of the accuracy of the trial kit for the detection of M., WV OPNREUMONI GE soccvorcsscreneseannesciwavareenwesessoenivencenvatavstans ori tees kuveueua tavepunseunceexsbonnaventies 31 4.2.2 Assessment of the stability of the trial kit for the detection of M hyopneumoniae
To define the limit of detection (LOD) of the Real-time PCR kit tested with
M hyopneumoniae reagent, standard samples containing Mí hyopneumoniae DNA plasmid with a concentration of 10° copies/uL were diluted to concentrations of 10°, 10*, 103, 102, 101, 10° copies/HL Each dilution was subjected to Real-time PCR three times, and the obtained results were tabulated in Table 4.1 The results showed that in all 3 repetitions for the FAM color channel signal amplification reaction, the reaction had the lowest concentration of 5 x 10! copies/reaction.
Table 4.1 Ct value of diluted MZ hyopneumoniae Plasmid DNA concentrations of 3 replicates
(copies/reaction) Rep 1 Rep 2 Rep 3 Mean Ct
The average Ct values from three replicates and corresponding DNA concentrations were utilized to generate a standard curve using Microsoft Office Excel
365 software The constructed standard curve is shown in Figure 4.2, with linear equation y = -3.5793x + 37.805 with Slope coefficient of -3.5793, correlation coefficient
R? = 0.9992, amplification efficiency (E%) = 90.02% According to Aaron Bivins
(2021) while amplification efficiency (E%) values often vary between samples, they are often highly repeatable for the same sample The ideal slope of a standard curve is -3.32,
28 signifying 100% amplification efficiency, though values ranging from -3.1 (E% 110%) to -3.58 (E% = 90%) are typical for real- time PCR testing using probe.
Figure 4.3 The standard curve represents the correlation between the Ct value and Log10 of the initial M hyopneumoniae DNA plasmid.
A standard curve should exhibit a strong linear correlation, characterized by an R² value between 0.98 and 1.00 The accuracy of this R² value is influenced by the precision of experimental procedures, equipment, and reagents used In this experiment, the standard curve achieved results with a slope, amplification efficiency (E%), and correlation coefficient (R’) all within acceptable limits Consequently, this standard curve equation is reliable for quantifying the initial number of M hyopneumoniae DNA copies present in the reaction.
The specificity of the test kit was thoroughly assessed using a diverse array of negative controls, which included various bacteria such as Erysipelothrix rhusiopathiae, Brordetella bronchiseptica, Haemophilus parasuis, Pasteurella multocida, Streptococcus Suis, Actinobacillus pleuropneumoniae, Escherichia coli, and Salmonella spp Additionally, viral controls included Porcine Circovirus type 2, African swine fever virus, and Porcine Reproductive and Respiratory Syndrome Virus, alongside a kit-specific negative control.
La ae ae a Se l a SR ee a 1 SS Í
Figure 4.4 Results of testing the specificity of the 7opSPEC@®
Mycoplasma hyopneumoniae qPCR KIT kit on the HEX color channel.
Fluorescence 8 ô > 3 8 |LuuuÍ i NTC vase) E.coli
Figure 4.5 Results of testing the specificity of the TopSPEC®
Mycoplasma hyopneumoniae qPCR KIT on the FAM color channel.
While Figure 4.3 confirmed successful amplification of the target
M hyopneumoniae DNA in the designated FAM channel, at the same time the amplification of the internal control (IC) in the HEX channel for all samples provided reassurance, verifying successful DNA extraction and proper functioning of the reagents and equipment, demonstrating the kit's ability to specifically detect
M.hyopneumomiae Figure 4.4, the E.coli control sample also exhibited amplification at Ct 32, indicating a non-specific reaction with the kit primers. This raises potential concerns about the kit's ability to accurately differentiate M.hyopneumoniae from other non-target agents in practical applications. Therefore, the specificity experiment was repeated with completely new E.coli isolates to verify whether the initial E coli sample was contaminated with the target agent M hyopneumoniae or there was a true non-specific binding of the primer. The results was presented in Table 4.2.
Table 4.2 Ct value results for the specificity test of the experimental kit with only
The specificity test results of the kit using E coli alone revealed amplification signals ranging from cycle 29.8 to cycle 32.74 across all three replicates This indicates non-specific binding of the kit's primers and probes to E coli genetic material The observed non-specific amplification underscores the need for further investigation into the primer pair's cross-reactivity to ensure reliable and accurate detection of M hyopneumoniae in practical applications.
4.2 Evaluation of the diagnostic performance of the trial kit for the detection of M hyopneumoniae in field samples
4.2.1 Evaluation of the accuracy of the trial kit for the detection of M. hyopneumoniae
An experiment was conducted to assess the accuracy of a trial kit for detecting Mycoplasma hyopneumoniae in lung tissue samples This evaluation involved three positive samples confirmed by the commercial Mycoplasma hyopneumoniae real-time PCR Kit (NZYtech) and three negative samples.
Table 4.3 Ct values of 3 replicates
Known samples as i nals a Rep 1 Rep 2 Rep 3 Mean Ct
The experiment demonstrated that all samples with a Ct value of less than 37 showed consistent amplification signals, with a standard deviation under 10% This indicates that the test kit is effective in detecting M hyopneumoniae DNA in field samples.
4.2.2 Assessment of the stability of the trial kit for the detection of M. hyopneumoniae
The stability of the Mycoplasma hyopneumoniae test kit was assessed using three samples confirmed positive for M hyopneumoniae with the NZYtech real-time PCR Kit The evaluation involved three repetitions of the test, and the corresponding Ct values of the replicates are detailed in Table 4.3.
Figure 4.6 Stability graph of the three replicates of
M hyopneumoniae positive samples generated using Microsoft Excel
The results in Figure 4.5 demonstrated the stability of the test kit in detecting
M hyopneumoniae based on the coefficient of variation (CV%) of the Ct values of three positive samples after three repetitions The experiment recorded the lowest CV% of 1.4% in sample P3 and the highest CV% of 2.2% in sample P2 In this study, the result indicates that the trial kit has high stability in detecting M hyopneumoniae. Nevertheless, the experiment should still be though necessitates further investigation with larger control sample to ensure reliable and accurate detection of M hyopneumoniae in practical settings.
4.2.3 Specificity, diagnostic sensitivity, positive predictive value, and negative predictive value of the trial kit
The experiment confirmed that there were no discrepancies in the results for M hyopneumoniae when compared to the control sample, resulting in 3 true negative and 3 true positive samples, with no false negatives or false positives recorded Consequently, the diagnostic specificity, sensitivity, positive predictive value, and negative predictive value of this test were all determined to be 100%.
Survey the kit's detection of M hyopneumoniae on field samples
An investigation was conducted to detect MZ hyopneumoniae in 50 field lung tissue samples using an experimental real-time PCR kit The findings revealed that 44% (22 out of 50) of the samples tested positive for M hyopneumoniae, while 56% (28 out of 50) were negative These results indicate that the trial PCR kit is effective in identifying the presence of M hyopneumoniae in various field sample matrices.
This study assessed a trial real-time PCR kit for the detection of M hyopneumoniae, showing strong agreement with earlier findings The insilico analysis of specificity also indicated a 100% concordance, consistent with the results reported by Tuan et al (2019) and Toan et al (2020) in their development of multiplex PCR primers.
M hyopneumoniae detection This outcome is considered standard in primer design, as mispriming, the non-specific amplification of unintended sequences, only occurs with poorly designed primers Mispriming leads to the amplification of unwanted sequences present in the template pool (Thornton and Basu, 2011).
In this study, the limit of detection was established at 50 copies per reaction across all three replicates In contrast, Lin et al (2023) reported a real-time PCR kit with a detection limit of 100 copies for M hyopneumoniae in the same target gene, P97 The slope coefficient was also noted.
Zhao et al (2018) created a real-time PCR kit that demonstrates excellent sensitivity, achieving an R² value of 0.992 and an amplification efficiency of 96.3% These results confirm the kit's high sensitivity and effective amplification, highlighting its potential for early diagnosis and detection of M hyopneumoniae, even at low abundance levels.
The specificity of the kit was tested against nine bacterial and viral species, showing non-specific primer binding to E coli within cycles 29.8 to 32.74 This misbinding may result from sequence homology between E coli and M hyopneumoniae (Smith et al., 2023) Additionally, the P97 gene encodes a highly conserved AAA+ ATPase protein essential for multiple cellular functions, such as protein unfolding and degradation (Iyer).
The sequence identity between MZ hyopneumoniae and E coli P97 genes is moderate, yet certain regions linked to protein-protein interactions or ATP binding may show greater homology This could result in non-specific primer binding in existing kits, underscoring the necessity of meticulously choosing primer sequences that focus on unique and specific areas.
M hyopneumoniae genome, particularly those with minimal sequence similarity to potential cross-reacting organisms like FE coli Several studies have explored the potential challenges associated with P97 gene-based primer design for
M hyopneumoniae detection Ma et al (2012) acknowledged the inherent difficulty in designing highly specific primers due to the conserved nature of the P97 gene across various bacterial species This further demonstrates the need for more extensive research and evaluation to improve primer sequences to minimize cross-reactivity with other species and achieve absolute specificity.
The kit showed precise detection of known control samples, achieving acceptable coefficients of variation (CV%) under 5% However, further research involving larger sample sizes is essential to confirm these results and improve their generalizability.
Ma et al (2012) assessed a real-time PCR kit with a sample size of 100, achieving a detection rate of 42%, which is similar to the 44% rate found in this study In contrast, Luo et al (2014) utilized a larger sample size of 200 and reported a higher detection rate of 65% with their real-time PCR kit These findings indicate that the detection rate in this study may be affected by sample size, underscoring the importance of conducting future research with larger and more diverse sample groups for comprehensive validation.
In conclusion, this study demonstrates the potential of the new kit for
M hyopneumoniae detection, aligning with previous reports in terms of specificity, sensitivity, and accuracy However, the observed non-specific amplification with E coli warrants further investigation, and the limited sample size necessitates validation with larger and more diverse samples to ensure reliable and accurate diagnosis 1n practical settings These findings emphasize the continuous need for improvement and validation 1n M hyopneumoniae detection methods.
CONCLUSION AND RECOMMENDATIONS
The study demonstrated that the tested Realttme PCR kit could detect
M hyopneumoniae at the limit of detection of 50 copies/reaction The equation of the standard curve was y = -3.5793x + 37.805, with a slope of -3.5793, an amplification efficiency (E%) of 90.02%, and a correlation coefficient (R?) of 0.999.
The amplification capacity demonstrates a commendable coefficient of variation (CV%) of 2.2% across three replicates However, under certain unspecified conditions, the primers exhibit weak and non-specific binding with F coli Consequently, additional research and enhancements to the kit are essential before it can be effectively utilized for diagnosing M hyopneumoniae in practical applications.
Further research is needed to improve the kit's specificity Continue to evaluate the experimental Real-time PCR kit on a wider range of sample matrices and larger sample sizes.
Dang Van Tuan, Lé Dinh Hai, và Nguyén Ba Hién (2019) đã nghiên cứu và thiết kế primer PCR multiplex nhằm phát hiện Mycoplasma hyopneumoniae, Mycoplasma hyorhinis và Mycoplasma bovis từ mẫu dịch phôi lợn Nghiên cứu này được công bố trên Tạp chí Khoa học và Công nghệ Việt Nam, góp phần quan trọng trong việc chẩn đoán và kiểm soát các bệnh do vi khuẩn Mycoplasma gây ra ở lợn.
Đỗ Xuân Dũng (2016) đã thực hiện một nghiên cứu sâu về cơ chế xâm nhiễm và gây bệnh của Mycoplasma hyopneumoniae trên tế bào phôi heo Luận án Tiến sĩ của ông được hoàn thành tại Đại học Nông nghiệp Hà Nội, đóng góp quan trọng vào hiểu biết về bệnh lý do Mycoplasma hyopneumoniae gây ra trong ngành chăn nuôi heo Nghiên cứu này không chỉ giúp xác định các cơ chế xâm nhiễm mà còn mở ra hướng đi mới trong việc phát triển các biện pháp phòng ngừa hiệu quả.
Lê Văn Toàn, Nguyễn Thị Phương Thảo, Phạm Thị Lan Anh, Nguyễn Thị Thu Hiền.
(2020) "Phát triển bộ primer PCR multiplex để chân đoán Mycoplasma hyopneumoniae va Mycoplasma bovis bằng phương pháp real-time PCR" Tap chi Khoa hoc va Công nghệ
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Appendix 1: Bioinformatics test results of the primer kit
>CP079799.1 Mesomycoplasma hyopneumoniae strain LH chromosome, complete genome product length = 90
Template S221 GỀNIè -sEsiblymez6athửsSi)(53ỏd9p5/2861500 02/086 222219
Template 22228) 6.55 G5 Gwe Saas ame SIE 222265
>CP058578.1 Mesomycoplasma hyopneumoniae strain ES-2L chromosome, complete genome product length = 90
Template 10009: 1g ci204niecử064060626isweeS-sue-Efe 218847
>CP034597.1 Mesomycoplasma hyopneumoniae strain F7.2C chromosome, complete genome product length = 90
Template 238366 newtieiew secs Sse Sewiales 238387
Template 7 Be hE ee Oe ee ae 238433
Appendix 1: Bioinformatics test results of the primer kit (continued 2)
>CP003802.1 Mycoplasma hyopneumoniae 7422, complete genome product length = 90
Template DANASS ioc icecye apeyeis ape pesados sms 241760
Template DATE2S edie sleiane diS.apkiane sIedleleleie wis 241806
>CP003131.1 Mycoplasma hyopneumoniae 168-L, complete genome product length = 99
Template TBS523) sư saisi6sieiessieviilisielgigieaslre 138545
>CP002274.1 Mycoplasma hyopneumoniae 168, complete genome product length = 90
Template ESBSO9 Si SeôicesiSs/esesseisiessisi where 138591
Appendix 1: Bioinformatics test results of the primer kit (continued 3)
>AED01 /244.1 Mycoplasma hyopneumoniae 7448, complete genome product length = 90
Template 222796 te 3awl 55/6 Gewese ces 222817
>AE017243.1 Mycoplasma hyopneumoniae J, complete genome product length = 90
Template PUSAGS diớgsefuel6:oe6ialefvlersoeeileisvee 215446
>AY512905.1 Mycoplasma hyopneumoniae strain ATCC 25934 adhesin gene, complete cds product length = 90
Appendix 1: Bioinformatics test results of the primer kit (continued 4)
>AY512904.1 Mycoplasma hyopneumoniae isolate 40689-2 adhesin gene, complete cds product length = 90
Template OF] cessed te elee os 016đ 895
>AY512903.1 Mycoplasma hyopneumoniae isolate 20868-B adhesin gene, complete cds product length = 90
Template BÍ? tu sateldildkows9sðisdne 895
>AY512902.1 Mycoplasma hyopneumoniae isolate 20735-2 adhesin gene, complete cds product length = 90
Template S28) acca orien eames seas 849
Template ODF? gi gayosolsooowade@epEtddxids 895
Appendix 1: Bioinformatics test results of the primer kit (continued 5)
>AY512900.1 Mycoplasma hyopneumoniae isolate 01-17704 adhesin gene, complete cds product length = 99
Template ĐO, giai Sea ers jHE:Ei2/9W6jsWxsdie 849
Template Cty va¿E2ểE6556S006xeuiabiter 895
>AY512898.1 Mycoplasma hyopneumoniae isolate IAF-DM9827 adhesin gene, complete cds product length = 99
>AY512895.1 Mycoplasma hyopneumoniae isolate IAF-669-00 adhesin gene, complete cds product length = 99
Appendix 1: Bioinformatics test results of the primer kit (continued 6)
>AY512894.1 Mycoplasma hyopneumoniae isolate IAF-464-01 adhesin gene, complete cds product length = 90
Template BZ caw pepereinnansnnpecacace ze deie: wpeilocent 849
>AY380564.1 Mycoplasma hyopneumoniae strain R659 nonfunctional P97 gene, complete sequence product length = 90
Template OSA) cvensntetoeayesttez304sdiel6tzsereerere 932
>U27294.1 Mycoplasma hyopneumoniae Mhp1 (mhp1) gene, complete cds product length = 90
Appendix 1: Bioinformatics test results of the primer kit (continued 7)
>AY957500.1 Mycoplasma hyopneumoniae ciliary adhesin protein P97 gene, complete cds product length = 90
>AF001398.1 Mycoplasma hyopneumoniae adhesin gene, complete cds product length = 90
>U50901.1 Mycoplasma hyopneumoniae ciliary adhesin protein P97 gene, complete cds product length = 90
Appendix 1: Bioinformatics test results of the primer kit (continued 8)
>AE017332.1 Mycoplasma hyopneumoniae 232, complete genome product length = 90
Template ZAR? giã 066 ss gee eR TREKS 226558
>AY380565.1 Mycoplasma hyopneumoniae strain F19 nonfunctional P97 gene, complete sequence product length = 99
Appendix 2: The results of 50 field samples were obtained when performing the Real-time PCR reaction.
No aS 18 Ct Values Results samples