Clinical Validation of Pre-amplification qPCR with DNA Free Extraction for the Rapid Detection of White Spot Syndrome Virus

doi:10.21203/rs.3.rs-1629186/v1

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Clinical Validation of Pre-amplification qPCR with DNA Free Extraction for the Rapid Detection of White Spot Syndrome Virus

https://doi.org/10.21203/rs.3.rs-1629186/v1

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WSSV has posed a serious threat to China and the global shrimp aquaculture industry. In order to diagnose WSSV early and prevent the spread and outbreak of WSSV, it is necessary to establish a highly sensitive WSSV diagnosis method suitable for shrimp farming sites. In this study, a pre-amplification qPCR assay with DNA free extraction was established, which was further compared with the universal qPCR assay to verify the clinical samples. The results show that the limits of detection (LOD) of pre-amplification qPCR were 1 copy / µl, and the LOD of universal qPCR were 10 copies / µl, respectively. It had good WSSV specificity and did not react with the other six viruses, including Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Hepatopancreatic Parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), Acute hepatopancreas necrosis disease (AHPND), Necrotizing hepatopancreatitis bacteria (NHPB), Decapod Iridescent Virus 1 (DIV1). A total of 36 clinical samples of WSSV-infected shrimp were diagnosed by pre-amplification qPCR with DNA free extraction and universal qPCR with DNA extraction. The diagnostic sensitivity and specificity were 97.22% (85.5% ~ 99.9%, 95% CI) and 100% (81.5% ~ 100%, 95% CI), respectively. The agreement Kappa value was 0.959 (0.879 ~ 1, 95% CI), and the analysis results were basically consistent. Eliminating the tedious steps of extracting DNA and using pre-amplified qPCR to detect WSSV in shrimp, it is a good choice for aquaculture farms.

White spot syndrome virus (WSSV)

Pre-amplification qPCR

DNA free extraction

Clinical Validation

Rapid detection

- Clinical Validation for the effect of pre-amplification qPCR with DNA free extraction for the rapid detection of white spot syndrome virus.

- The pre-amplification qPCR assay was fast, sensitive, and easy to operate, which was more suitable for the detection of white spot syndrome virus in shrimp culture site.

White spot syndrome virus (WSSV) is an important pathogen endangering shrimp culture (Lei et al. 2017). White spot disease (WSD) caused by the virus have brought great harm and serious economic losses to shrimp breeding industry in China and all over the world (Millard et al. 2021). In 1921, WSD was first discovered in China Taiwan (Zhu et al. 2019), and have spread to other Asian countries (Cui et al. 2020, Boonyakida et al. 2021). In 1995, WSD was also found in a shrimp farm in southern Texas (Sanchez-Paz 2010). The genome of WSSV are huge, and the structural proteins mainly include nucleocapsid protein, envelope protein and envelope protein (Li et al. 2019, Lu et al. 2019, Boonyakida et al. 2020, Shine et al. 2020). The infection mechanism of WSSV are complex (Liu et al. 2021). WSSV can spread in a wide range through the water area, with the characteristics of wide host range, strong infection ability and high mortality rate. Crustacean aquatic animals such as shrimp and crab can be infected (Ding et al. 2015). The mortality of aquatic animals are more than 70% within 3 ~ 10 days after infection, and that of shrimp are almost 100% (Wen 2017). Due to the rapid and vast spread of WSSV in China, the aquaculture industry has responded promptly to outbreaks, and the government has issued emergency plans, such as expert consultation and target surveillance programs. The world organization for animal health (OIE) and the network of Aquaculture Centers in Asia Pacific (NACA) have listed WSD as a compulsory notification of aquatic animal diseases (Xie & Wang). China has included it as a Class II animal epidemic disease in the list of quarantine diseases of imported animals of the people's Republic of China (2014). So far, there is no effective drug to treat WSSV, hence, rapid detection and monitoring on the breeding site are the key measure to control virus transmission (Verbruggen et al. 2016).

Antibody dependent immunological assays are fast and have been used for rapid screening of WSSV (Kulabhusan et al. 2017, Tang et al. 2018). However, the success of immunoassay of viruses depends on the high affinity and specificity of antibodies (Mangal et al. 2016). Immune antibodies usually have low sensitivity and specificity to some mutant viruses, and are easily affected by matrix interference, which will limit the stable expression of target virus antigen, and then affect the accuracy of detection (Su et al. 2021). Polymerase chain reaction (PCR) are typical and advanced nucleic acid amplification technology, which have the advantages of simplicity and high efficiency (Tang et al. 2013, Thamizhvanan et al. 2019). Nested PCR have high specificity and sensitivity, and are used to detect WSSV (Chang et al. 2020). However, nested PCR assays are cumbersome and difficult to popularize. Real-time quantitative PCR (qPCR) have good specificity and sensitivity, and have been applied to detect WSSV (Meng et al. 2010, Jang et al. 2011). Loop mediated isothermal amplification (LAMP) based on thermostatic amplification have been used for rapid detection of WSSV in the field, and its simple equipment, faster speed and amplification of targets at constant temperature (Li et al. 2015, Wu 2019, Gong et al. 2021). However, LAMP primers (4–6 primers are required) are more difficult to design than qPCR primers. Compared with qPCR, LAMP assays have slightly higher false positive rate and slightly lower sensitivity (Xia et al. 2014). Recombinase polymerase amplification (RPA) and recombinase mediated amplification (RAA) can complete the rapid amplification of nucleic acid at 37 ℃ for about 30 minutes, which have been used in the study of rapid detection of WSSV (Huang et al. 2022). However, the stability of RPA and RAA are poor, and the reagent price are relatively expensive. In contrast, qPCR assays are more widely and stably applied to the detection of WSSV (Qiu et al. 2018), and its reagents are easier to obtain. Its specificity and sensitivity are higher than those of LAMP, RPA and RAA assays, which are more suitable for the analysis of low concentration virus clinical samples. DNA extraction in qPCR detection process are cumbersome and time-consuming, which limits the application of qPCR assays in the field. Studies have shown that pre-amplified qPCR can overcome the problem of insufficient copy number of target gene in the reaction (Korenkova et al. 2015). In this study, we aim to establish a pre-amplified qPCR assays for detecting WSSV, with DNA free extraction from clinical shrimp samples, so as to reduce the operation links, and verify the effect of pre-amplified qPCR in detecting clinical samples compared with universal qPCR. This paper attempts to find a convenient and highly sensitive method for detecting WSSV in shrimp culture site.

Clinical samples and WSSV standard plasmid

In total, 54 Penaeus vannamei samples were collected from four shrimp farms in Liaoning Province (China). WSSV-infected dead shrimp samples were obtained from Guangzhou Double Helix Gene Technology Co., Ltd. (Guangzhou, China). A total of 6 other non-WSSV-infected diseased shrimp samples were obtained from Beijing Lambrui Biotechnology Co., Ltd (Beijing, China) and were used for the specificity assay in this study, including samples infected with Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Hepatopancreatic Parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), Acute hepatopancreas necrosis disease (AHPND), Necrotizing hepatopancreatitis bacteria (NHPB), Decapod Iridescent Virus 1 (DIV1). All of the samples for the specificity assay were validated by sequencing and a published PCR assay. The full sequence of the WSSV VP664 gene (GenBank accession number MN481520.1) was inserted into the commercial vector pMD19 (TaKaRa Co., Ltd., Dalian, China) to construct a standard plasmid (1.0×107 copies/µl). The synthesis of the standard plasmid was completed by TaKaRa Co., Ltd. (Dalian, China).

Clinical samples with DNA free extraction

Took 0.1g ~ 0.2g of shrimp tissue samples, were placed in a grinding tube containing phosphate buffer solution (PBS). After 6000 r/min shaking grinding for 45 s, about 10% tissue homogenate ware prepared and centrifuged at 5000 r/min for 5 min. Took 5 ~ 20µl supernatant and added 100µl MightyPrep reagent (Code 9182, TaKaRa Co., Ltd., Dalian, China), mixed by oscillator. Heated at 95℃ for 10 min; centrifugation at 12000 r/min for 2 min. The supernatant ware directly used for pre-amplification qPCR assays.

Clinical samples DNA extraction

DNA was extracted from 5 ~ 20 µl supernatant prepared from the above operation process, with magnetic bead virus DNA extraction kit (DP438-T2K, Tiangen Biochemical Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The DNA templates were used for universal qPCR assays.

qPCR primers and probes design

The WSSV nucleocapsid protein VP664 gene (GenBank No. MN481520.1) ware selected to design primers targeted to specific conserved DNA fragments in this work. The forward and reverse primers and probes were designed for qPCR using Primer Express software 5.0 (Applied Biosystems, Foster City, CA, USA). Forward primer, 5'-CGATCTTGGAAAGGTTATC-3'; Reverse primer, 5'- GAGCCTAGTCTATCAATCA-3'; Probe, FAM-5'-CGGCACCATCGCTGAATCTG-3'-BHQ1. The primers and probe were synthesized from TaKaRa Co., Ltd. (Dalian, China).

Universal qPCR assay

Universal qPCR assays ware performed on CFX96 (BIO-DL, USA). The reactions were prepared as a 25 µl reaction volume containing 12.5 µl probe qPCR mix containing enzyme (Code391A, TaKaRa Co., Ltd., Dalian, China), 0.5 µl (0.2 µM) forward primers, 0.5 µl (0.2 µM) reverse primers, 1 µl (0.4 µM) probe and 2 µl extracted DNA of samples templates. Universal qPCR thermal cycling program ware: 30 s at 95℃, followed by 40 cycles of 5s at 95℃ and 30s at 60℃; Fam fluorescence signals were collected at 60℃.

Pre-amplification PCR Assay

Pre-amplification qPCR assays ware performed on CFX96 (BIO-DL, USA). The reactions were prepared as a 25 µl reaction volume containing 12.5 µl probe qPCR mix containing enzyme (Code391A, TaKaRa Co., Ltd., Dalian, China), 0.5 µl (0.2 µM) forward primers, 0.5 µl (0.2 µM) reverse primers, 1 µl (0.4 µM) probe and 2 µl supernatant with DNA free extraction. Pre-amplification qPCR thermal cycling program ware: first, pre-amplification step (15 cycles of 10 sec at 95℃ and 10 sec at 50℃), and Fam fluorescence signal ware not collected; next, 1 min at 95℃; followed by 35 cycles of 10 sec at 95℃ and 30 sec at 55℃; Fam fluorescence signals were collected at 55℃.

Analytical Sensitivity and Specifificity of pre-amplification qPCR and universal qPCR

To determine the limit of detection for pre-amplification qPCR and universal qPCR, serial dilutions of WSSV standard plasmid for concentration including 1.0×10⁷, 1.0×10⁶, 1.0×10⁵, 1.0×10⁴, 1.0×10³, 1.0×10², 1.0×10¹, 1.0×10⁰ copies/µl were prepared. Each concentration was assayed in three replicates by pre-amplification qPCR and universal qPCR. The analytical specificity of WSSV by pre-amplification qPCR and universal qPCR were evaluated in WSSV-infected diseased shrimp samples and other pathogens with similar viral symptoms (IHHNV, HPV, EHP, AHPND, NHPB, DIV1). Healthy shrimp were used as negative control.

Comparison of pre-amplification qPCR with DNA free extraction and universal qPCR with DNA extraction using clinical samples

54 Penaeus vannamei clinical samples were collected from four shrimp farms in Liaoning Province (China). To explore the clinical performance of pre-amplification qPCR assay with DNA free extraction in the detection of clinical specimen. The performance of pre-amplification qPCR assay with DNA free extraction were compared to that of universal PCR assay with DNA extraction. The degree of agreement between pre-amplification qPCR with DNA free extraction and universal qPCR with DNA extraction assay results were measured with kappa value by using MedCalc software (MedCalc Software bvba, Ostend, Belgium).

Statistical Analysis

Data in this study were presented as mean ± standard deviation. For the determination of WSSV for pre-amplification qPCR assay and universal qPCR assay analytical sensitivity, a semi-log regression analysis (PRISM, Graphpad Software Inc., San Diego, CA, United States). The probit regression analysis using MedCalc Software (MedCalc Software bvba, Ostend, Belgium) was performed for the pre-amplification qPCR assay and universal qPCR assay at a 95% probability level.

Sensitivity and Specificity of WSSV pre-amplification qPCR and universal qPCR

The sensitivities of the newly established pre-amplification qPCR and universal qPCR assays were evaluated using continuous dilutions of the WSSV standard plasmid DNA. The results indicated that the pre-amplification qPCR and universal qPCR assays could efficiently detect low levels of WSSV (Fig. 1). The minimum concentration of 100% positive results was defined as the limit of detection (LOD). The LOD of pre-amplification qPCR assay was 1 copies/µl (Fig. 1A, 1B), and the LOD of universal qPCR assay was 10 copies/µl (Fig. 1C, 1D). The specificities of the pre-amplification qPCR and universal qPCR assays of WSSV were assessed using clinical shrimp samples positive for WSSV, and with shrimp samples containing 6 other viruses, including IHHNV, HPV, EHP, AHPND, NHPB, DIV1 and healthy shrimp. The results indicated that only the WSSV positive clinical samples completed the index amplification; the 6 other viral samples and healthy shrimp have no amplification curves and were negative (Fig. 1E, 1F). Thus, the established pre-amplification qPCR and universal qPCR assays WSSV detection method have good specificity.

[Figure 1 near here]

Performance of WSSV pre-amplification qPCR with DNA free extraction on clinical samples and its comparison with universal qPCR with DNA extraction testing

The purpose of this study was to detect WSSV by pre-amplification qPCR with DNA free extraction in shrimp farms. In order to evaluate the practical application of WSSV pre-amplification qPCR with DNA free extraction in this work, 54 clinical samples were tested and the results were compared with that universal qPCR with DNA extraction (Table 1). By the detection of universal qPCR with DNA extraction, 36 samples were confirmed as WSSV DNA positive (Ct value, ranging from 18.32 to 38.79) while 18 as negative (Ct value, undetermined). By the detection of pre-amplification qPCR with DNA free extraction in this work, 35 samples were identified as WSSV DNA positive (Ct value, ranging from 11.61 to 23.19) and 19 as negative (Ct value, undetermined). Linear correlation analysis revealed that, as the Ct value of universal qPCR with DNA extraction increased, the Ct value by pre-amplification qPCR with DNA free extraction present a growing trend correspondingly (Fig. 2). Agreement analysis based on the detection of clinical samples showed that the kappa value between pre-amplification qPCR with DNA free extraction and universal qPCR with DNA extraction was 0.959 (0.817∼1, 95% CI). Additionally, in comparison to universal qPCR with DNA extraction, the specificity and the sensitivity of pre-amplification qPCR assay with DNA free extraction for identification of WSSV were 100% (81.5%∼100%, 95% CI) and 97.22% (85.5%∼99.9%, 95% CI), respectively, indicating an excellent diagnostic agreement between pre-amplification qPCR with DNA free extraction and universal qPCR with DNA extraction (Table 2).

[Table 1 near here]

Table 1 Detection of WSSV by Pre-amplification qPCR with DNA free extraction in clinical samples.

Sample ID	qPCR with DNA extraction		Pre-amplification qPCR with DNA free extraction
Sample ID	Ct value	Result	Ct value	Result
P1	29.37	+	16.61	+
P2	36.63	+	22.19	+
P3	29.58	+	16.25	+
P4	20.53	+	16.59	+
P5	19.44	+	17.41	+
P6	26.37	+	18.61	+
P7	30.78	+	19.83	+
P8	27.27	+	21.45	+
P9	32.08	+	19.54	+
P10	26.25	+	19.73	+
P11	26.45	+	14.67	+
P12	38.79	+	Undet.^a	̶
P13	25.75	+	12.57	+
P14	18.32	+	13.43	+
P15	29.37	+	16.27	+
P16	32.23	+	19.31	+
P17	19.54	+	12.81	+
P18	32.47	+	18.71	+
P19	30.41	+	17.70	+
P20	23.28	+	14.73	+
P21	25.53	+	15.33	+
P22	26.27	+	13.78	+
P23	32.16	+	20.39	+
P24	25.16	+	13.41	+
P25	33.37	+	23.19	+
P26	24.31	+	13.11	+
P27	20.35	+	11.61	+
P28	25.39	+	13.45	+
P29	28.71	+	16.43	+
P30	26.42	+	14.51	+
P31	28.34	+	13.76	+
P32	24.85	+	13.63	+
P33	28.71	+	18.49	+
P34	26.46	+	14.16	+
P35	26.37	+	13.72	+
P36	24.85	+	12.23	+
N1	Undet.	̶	Undet.	̶
N2	Undet.	̶	Undet.	̶
N3	Undet.	̶	Undet.	̶
N4	Undet.	̶	Undet.	̶
N5	Undet.	̶	Undet.	̶
N6	Undet.	̶	Undet.	̶
N7	Undet.	̶	Undet.	̶
N8	Undet.	̶	Undet.	̶
N9	Undet.	̶	Undet.	̶
N10	Undet.	̶	Undet.	̶
N11	Undet.	̶	Undet.	̶
N12	Undet.	̶	Undet.	̶
N13	Undet.	̶	Undet.	̶
N14	Undet.	̶	Undet.	̶
N15	Undet.	̶	Undet.	̶
N16	Undet.	̶	Undet.	̶
N17	Undet.	̶	Undet.	̶
N18	Undet.	̶	Undet.	̶

^aUndet., no amplification detected.

[Figure 2 near here]

[Table 2 near here]

Table 2

Diagnostic performance comparison between pre-amplification qPCR with DNA free extraction and universal qPCR with DNA extraction.
clinical samples	Universal qPCR with DNA extraction		Pre-amplification qPCR with DNA free extraction		Performance characteristics (%)
	Positive	Negative	Positive	Negative	Sensitivity	Specificity
Positive	36	0	35	1	97.22% (85.5%∼99.9%, 95% CI)	100% (81.5%∼100%, 95% CI)
Negative	0	18	0	18
Total	54		54
Agreement Kappa value: 0.959 (0.879∼1, 95% CI)

WSSV have posed a serious threat to China and the global shrimp breeding industry (Millard et al. 2021). Considering the prevention of heavy losses in shrimp farming, it is very important to establish a simple and sensitive diagnostic method for early diagnosis of WSSV in shrimp farms. Traditional cell culture for virus detection are expensive and time-consuming. The time required for ELISA assay are also longer than that for nucleic acid assay(Ozer et al. 2020). qPCR assays are the "gold standard" for the detection of most viruses. Compared with LAMP, RPA and RAA assays, qPCR assays are more suitable for the analysis of low concentration virus clinical samples. Based on these considerations, in this study, we have developed a pre-amplified qPCR assay, which were suitable for DNA free extraction and can be used for rapid detection of shrimp clinical samples in farms. The pre-amplified qPCR assay based on DNA free extraction showed good sensitivity, specificity and accuracy for clinical samples. In terms of sensitivity, pre-amplified qPCR assay through 15 cycles of accelerated pyrolysis, did not collect fluorescence signals and accumulated templates, and have higher sensitivity than universal qPCR assay and are more suitable for the detection of low concentration viruses.

This is the first study to establish a pre-amplified qPCR assay with DNA free extraction for WSSV identification. In comparison, indicating an excellent diagnostic agreement between pre-amplification qPCR with DNA free extraction and universal qPCR with DNA extraction. Eliminating the tedious steps of extracting DNA, using pre-amplified qPCR to detect trace pathogens in aquatic products is a good choice for aquaculture farms. It can provide reference value for early monitoring and control of WSSV in aquaculture industry.

Ethical Approval and Consent to participate

Not applicable.

Human and Animal Ethics

Not applicable.

Consent for publication

Not applicable.

Availability of supporting data

The data that support the fndings of this study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no conflict of interests.

Funding

This work was supported by “Rejuvenating Liaoning Talents Plan” of Liaoning Province (XLYC2002106) and (2019CT09), China.

Authors' contributions

Chao Ma and Lili Yang finished experiment operation. Chao Ma and Zhuo Tian wrote the main manuscript text and prepared figures and tables. Jijuan Cao completed the fund acquisition, supervision and editing. All authors reviewed the manuscript. Chao Ma and Zhuo Tian contributed equally to this work.

Acknowledgements

This work was supported by “Rejuvenating Liaoning Talents Plan” of Liaoning Province (XLYC2002106) and (2019CT09), China. We thank Dr. Huang Zhang for performing the validation of clinical samples in Guangzhou Double Helix Gene Technology Co., Ltd. (Guangzhou, China).

Authors' information

Chao Ma ^a,^† , Zhuo Tian ^b,^† , Lili Yang^a, Jijuan Cao ^a,*

^aKey Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, Dalian Minzu University, Dalian, China ;

^b Dalian Customs Technology Center, Dalian, China ;

^*Corresponding author, Email: [email protected] ;

^†These authors contributed equally to this work.

ORCID:

Chao Ma: 0000-0001-9703-4693

Zhuo Tian: 0000-0002-4765-2992

Lili Yang: 0000-0002-9300-7126

Jijuan Cao: 0000-0002-7365-9557

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No competing interests reported.

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Editorial decision: Major revision
27 May, 2022
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Clinical Validation of Pre-amplification qPCR with DNA Free Extraction for the Rapid Detection of White Spot Syndrome Virus

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Version 1

Abstract

Figures

Highlights

Introduction

Materials And Methods

Clinical samples and WSSV standard plasmid

Clinical samples with DNA free extraction

Clinical samples DNA extraction

qPCR primers and probes design

Universal qPCR assay

Pre-amplification PCR Assay

Analytical Sensitivity and Specifificity of pre-amplification qPCR and universal qPCR

Statistical Analysis

Results And Discussion

Sensitivity and Specificity of WSSV pre-amplification qPCR and universal qPCR

Conclusions

Declarations

Ethical Approval and Consent to participate

Human and Animal Ethics

Consent for publication

Availability of supporting data

Competing interests

Funding

Authors' contributions

Acknowledgements

Authors' information

References

Additional Declarations

Status:

Version 1