Step Polymerase Chain Reaction Research Articles

Development of a 1-step multiplex PCR assay for the detection of S. Enteritidis, S. Pullorum, S. Typhimurium, and S. Infantis associated with poultry production

Salmonellosis in poultry is detrimental to the advancement of the breeding industry and poses hazards to human health. Approximately 2,600 Salmonella varieties exist, among which S. Enteritidis, S. Pullorum, S. Typhimurium, and S. Infantis are prevalent serotypes in the poultry industry in recent years. They can also infect humans by contaminating poultry eggs and meat. Therefore, identifying these serotypes is crucial for successful preventive and control interventions. The White-Kauffmann-Le Minor scheme is time-consuming and requires expensive reagents. Whole-genome sequencing (WGS) and other molecular biology techniques require skilled technical staff. In comparison, the polymerase chain reaction (PCR) is more accurate, rapid, and inexpensive, thus proving suitable for widespread application in the poultry industry. Here, we selected 4 specific primers: lygD, mdh, ipaJ, and SIN_02055, which correspond to detecting S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Infantis, respectively. They were integrated into a 1-step multiplex PCR method. We optimized the PCR method by utilizing specificity test results to determine the optimal annealing temperature (57°C). The PCR method exhibited excellent sensitivity for genomic DNA and bacterial cultures. We used the developed method to determine 157 clinical Salmonella isolates from various stages of the poultry production chain. The results aligned with serotype data generated via WGS analysis, demonstrating the method's excellent accuracy. In conclusion, this study developed a 1-step multiplex PCR method that simultaneously identifies S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Infantis, allowing routine mass detection in the grass-root poultry industry.

HIV and Malaria co-infection and the impact of viral load and HAART usage on the development of Plasmodium falciparum Artemisinin and Lumefantrine resistant genes in Nnewi, Anambra State, Nigeria

Background: Human Immunodeficiency virus (HIV) and malaria co-infection poses a serious health threat in sub-Saharan Africa and other endemic countries. Highly active anti-retroviral therapy (HAART) is currently used to suppress viral loads. Methods: Blood samples collected from 400 participants comprising 200 HIV sero-positive and 200 sero-negative individuals was added to EDTA sample containers. Malaria parasitemia was evaluated using standard parasitological techniques followed by PCR techniques using the Quick Load One Taq One Step Polymerase Chain Reaction (PCR) for characterization of species of Plasmodium and resistant studies using specific primers. HIV viral load estimation was done using COBAS® TaqMan® Analyzer. Results: Malaria has prevalent rate of 22.75% in the study population, while the prevalence of malaria infection among the HIV sero-positive and sero-negative is 77.0% and 23% respectively. Socio-demographic factors had no significant association with the development of resistant genes. HAART exposed individuals had prevalence of PfK13 (6.9%) and Pfmdr-1 (20.8%). Viral load was significantly related with the development of resistant genes (100%) and (86.1%) for PfK13 and Pfmdr-1 respectively. Conclusion: Unsuppressed viral load in HIV sero-positive individuals heightens the prevalence of malaria parasitaemia and increases the chances of possible emergence and spread of PfK13 and Pfmdr-1 genes.

Development and evaluation of a ligation-free sequence-independent, single-primer amplification (LF-SISPA) assay for whole genome characterization of viruses

Molecular identification and characterization of novel or re-emerging infectious pathogens is critical for disease surveillance and outbreak investigations. Next generation sequencing (NGS) using Sequence-Independent, Single-Primer Amplification (SISPA) is being used extensively in sequencing of viral genomes but it requires an expensive library preparation step. We developed a simple, low-cost method that enriches nucleic acids followed by a ligation-free (LF) 2-step Polymerase Chain Reaction (PCR) procedure for library preparation. A pan-chimeric universal primer (JS15N14) containing 15 nucleotides with a random tetradecamer (14N) attached to the 3′-end was designed. The complimentary primer (JS15) was used for nucleic acid enrichment in a first round PCR. A second PCR was designed to create Illumina sequencer-compatible sequencing-ready libraries for NGS. The new LF-SISPA protocol was tested using six RNA and DNA viral genomes (10.8–229.4 kilobases, kb) from an ATCC virome nucleic acid mix (ATCC® MSA-1008™) followed by analysis using One Codex, an online identification tool. In addition, a human stool sample known to be positive for norovirus GII was sequenced, and de novo assembly was performed using the Genome Detective Virus Tool allowing for near complete genome identification in less than 24 h. The LF-SISPA method does not require prior knowledge of target sequences and does not require an expensive enzymatic library preparation kit, thereby providing a simple, fast, low-cost alternative for the identification of unknown viral pathogens.

Exploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4

BackgroundStudies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls.ResultsThe number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups.ConclusionsDifferences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

Evidence of occurring alfalfa mosaic virus in potato plants in Assam, India.

Alfalfa mosaic virus (AMV, family Bromoviridae, genus Alfamovirus) has an extensive host range. The reports of AMV available in India were dated far back as 1979 and 1981 found in alfalfa and brinjal crops respectively. In January 2019, field surveys were conducted for viral diseases infecting potato in Sonitpur and Jorhat districts of Assam state of India. Severe yellow mosaic or calico pattern symptom, consistent with infection with AMV were observed with an incidence of approximately 25% of the plants found in farmer's fields. Sixty different symptomatic leaf samples including those associated with AMV observed were collected at random and were analysed to detect the presence of AMV. Leaf samples were frozen in liquid nitrogen and total RNA extracted from them were analyzed by one step polymerase chain reaction to detect the presence of AMV reported in potato inducing similar symptoms using a specific pair of primers for coat protein gene. An expected amplicon size of 351bp was observed in 70% of the symptomatic leaf samples when the PCR products were analyzed on a 1.2% agarose gel. The PCR product for one sample each from the surveyed districts was eluted, purified and sequenced. The sequence results obtained were compared with those deposited in GenBank database. Blastn analysis of the sequenced isolates submitted to GenBank revealed nucleotides similar to AMV Iran isolate sequences. To our knowledge, thisis the first report of AMV infecting potato in India.

Novel molecular diagnostic technique for detecting the different species of Plasmodium

BackgroundSensitive diagnostic techniques are needed for timely detection of malaria parasite and disease control. Molecular diagnostic techniques involving Polymerase chain reaction (PCR) with 18 s rRNA as a known diagnostic target with an overall sensitivity of 10 parasites per microliter is used as a gold standard. Till date, no attempt has been undertaken to develop a technique for the identification of four Plasmodium species in a single step PCR combined with restriction digestion with enzymes. MethodPlasmodium species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays have been developed, based on RFLP of amplified PCR product of mitochondrial gene as a target. This approach identifies Plasmodium species in two steps involving amplification of mitochondrial (Mt) gene by PCR followed by digestion with restriction enzymes. ResultA total of 36 clinical samples were subjected to PCR-RFLP for the diagnosis and detection of malaria parasites targeting mitochondrial gene (Mt). The findings of the method were compared with gold standard methods (Microscopy, RDTs and Nested PCR) and was able to detect mixed infection with a sensitivity of 100% and specificity of 93.8% with respect to nested PCR. The results obtained by PCR-RFLP were validated with Sanger sequencing (n = 32) and were found to be consistent with the method. ConclusionThis method identifies and distinguishes four species of human malaria parasite namely P. falciparum (Pf), P. vivax (Pv), P. malariae (Pm) and P. ovale (Po) in approximately 4 h. To overcome and address PCR difficulties, continuous efforts are needed for the development of newer diagnostic techniques.

An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies.

BackgroundThe use of environmental DNA for species detection via metabarcoding is growing rapidly. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the 2 most important risks of environmental DNA use: sample contamination and taxonomic misassignment. These risks arise from the need for polymerase chain reaction (PCR) amplification to detect the trace amounts of DNA combined with the necessity of using short target regions due to DNA degradation.FindingsOur high-throughput workflow minimizes these risks via a 4-step strategy: (i) technical replication with 2 PCR replicates and 2 extraction replicates; (ii) using multi-markers (12S,16S,CytB); (iii) a “twin-tagging,” 2-step PCR protocol; and (iv) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases. Because annotation errors in the reference sequences can result in taxonomic misassignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in >50% of sequences being deleted from public reference databases, owing to (i) limited overlap between our target amplicon and reference sequences, (ii) mislabelling of reference sequences, and (iii) redundancy. Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an invertebrate-derived DNA dataset from 1,532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criterion we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples.ConclusionsOur metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider use of environmental and invertebrate-derived DNA, which is turning into a valuable source of ecological and conservation information on tetrapods.

A new method to quantify atmospheric Poaceae pollen DNA based on the trnT-F cpDNA region

Abstract Background Pollen, mold spores, bacteria and viruses are the main biological substances in the atmosphere causing allergic symptoms and disease. Distinguishing pollen and spores is quite time consuming and requires a trained expert. There is a different approach to identification of these substances such as microscopic analysis. However, DNA based identification of these is becoming popular recently. Objective We evaluated the correlation between the quantity of DNA, which was amplified using trnT-F cpDNA specific primers in samples obtained from a high volume air sampler (HVAS), and concentration of Poaceae pollen collected with a Burkard trap. Materials and methods Here, we present a method for identifying and quantifying airborne Poaceae pollen using a single step polymerase chain reaction (PCR) technique. Forty daily air samples were collected by HVAS. The method was optimised using two different methods (M1 and M2) and the trnT-F cpDNA region was amplified using a Poaceae specific primer pair. The correlation between the quantity of DNA and pollen concentration was tested using R statistical programming language. Results Although a significant correlation was obtained between the M1 and M2 methods (R2=0.655, p<0.01), the M2 method was more correlated with pollen concentration. The correlation between pollen and DNA content changed due to episodes that were observed during the pollen season. DNA concentrations from the PCR data were significantly correlated with pollen concentrations determined by light microscopy (R2=0.767, p<0.01) in episode II using the M2 method and during the entire season (R2=0.469, p<0.01) using M2. Conclusions The M2 method correctly identified Poaceae pollen in mixed air samples from Zonguldak Province. The non-coding trnT-F cpDNA region was used for the first time in aerobiological samples to identify Poaceae pollen. Use of this method that does not require DNA extraction may be a crucial step for real-time pollen monitoring devices to be developed in the future. The correlation strength between pollen and amplified DNA content could be improved using a sampler that has a lower absorption rate, and a more sensitive technique, such as qPCR.

Exosomes molecular diagnostics: Direct conversion of exosomes into the cDNA for gene amplification by two-step polymerase chain reaction.

Exosomes are cell derived lipid nanoparticle with a size of 30–100 nm in diameter, found in almost all biological fluids. The composition of the exosomes is mainly lipid, proteins, RNA, DNA, and non-coding RNAs. Currently, most available methods and commercial kits for exosomal-RNA (Exo-RNA) isolation have limitations and shortcomings. Small starting volume of exosomes and the use of extraction/filtration columns results usually insufficient yield of exosomal RNA after isolation. The majority of RNA contained in purified exosomes range in size from 15–500 nucleotides. Some RNA isolation kits are well suited for small RNA transcripts isolation but larger mRNA transcripts are hard to detect. For all of the kits, the cost prize per sample analyzed is very high. Our current method provides a novel way for direct conversion of exosomes into cDNA synthesis (Exo-cDNA) and subsequent gene detection by polymerase chain reaction (PCR). This method has several advantages compared to established available kits. No extraction column is utilized in this procedure which means total recovery of exosomal RNA with maximal yield. In addition, this method is fast and uses a minimal amount of lab supplies, thereby reducing the overall working costs. Our findings suggest that direct conversion of exosomes into cDNA and subsequent gene amplification by two step PCR is a most efficient and reproducible technique. This novel method can be applied to and is useful to advance molecular research of exosomes by solving the problem of low molecular yields.

Detection of viruses in the exotic shrimp Penaeus vannamei Boone, 1931 cultured in India

Pacific white shrimp Penaeus vannamei Boone, 1931, is a recently introduced species in India. P. vannamei samples, collected from various shrimp farms of Karnataka, India were subjected to polymerase chain reaction (PCR) based detection of whitespot syndrome virus (WSSV), hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), taura syndrome virus (TSV) and infectious myonecrosis virus (IMNV). Out of the 81 shrimp samples analysed, 41 samples (50.6%) were found positive for WSSV and four (4.9%) were positive for IHHNV. Among 41 WSSV positive samples, 20 (48.7%) samples were found positive for WSSV by 1st step PCR, while the remaining 21 (51.2%) samples were positive by nested PCR. WSSV positive samples were further confirmed by dot blot hybridisation assay. However, clinical signs/disease symptoms were not observed in any of the shrimp samples tested positive for the viruses.

Comparative evaluation of the GP5 +/6 +, MY09/11 and PGMY09/11 primer sets for HPV detection by PCR in oral squamous cell carcinomas

The aim of this study was to evaluate the use of GP5+/6+, MY09/11 and PGMY09/11 primer sets for the detection of human papillomavirus (HPV) DNA by single step polymerase chain reaction (PCR) and nested PCR in formalin-fixed and paraffin-embedded (FFPE) tissues from oral squamous cell carcinomas (OSCCs). DNA extracted from FFPE tissues were tested for amplification of the human beta globin gene with PCO3/4 primers. Positive samples for this gene were tested for HPV DNA using single step PCR with GP5+/6+, MY09/11 and PGMY09/11 primer sets. All negative samples at single step PCR with MY09/11 and PGMY09/11 were subjected to a further PCR with GP5+/6+ primers using the non-amplified product in the previously reactions (nested PCR) as samples. Among 26 samples, 23 were positive for the human beta globin gene and were considered viable for HPV DNA detection by PCR. Single step PCR with GP5+/6+ and MY09/11 primers and MY/GP+ nested PCR did not amplify HPV DNA in any samples. PGMY09/11 primers detected HPV DNA in 13.0% of OSCC cases and this rate was raise to 17.4% with the use of PGMY/GP+ nested PCR. According to our results the PGMY/GP+ nested PCR is the most appropriate primer set for the detection of HPV DNA using FFPE samples from OSCC.

Authentication of goat (Capra hircus) meat using PCR amplification of mitochondrial cytochrome b gene

A highly specific single step polymerase chain reaction (PCR) is described for the detection of goat (Capra hircus) meat. A PCR assay was successfully optimized for amplification of 617-bp DNA fragment extracted from goat meat using designed species-specific primer pairs based on mitochondrial cytochrome b gene. The optimized PCR assay was subsequently validated for its specificity with DNA extracted from cattle, buffalo, sheep, goat and pig. PCR amplification of target DNA with goat-specific primers was repeated 10 times, with consistent results observed. The specificity of goat-specific PCR provides a valuable tool for identification of goat meat and to avoid its fraudulent substitution and adulteration.

One step PCR for detection of Staphylococcus aureus specific sequence gene and mecA gene in Northwestern Nigerian hospitals

Methicillin – resistant Staphylococcus aureus (MRSA) has been noted as one of the main pathogen of public health importance. Detection of the mec A gene by polymerase chain reaction (PCR) is the gold standard for identifying methicillin-resistant Staphylococcus aureus.

K-RAS GENE MUTATIONS AT CODON 12 AND 13 IN PANCREATIC CANCER AND CHRONIC PANCREATITIS PATIENTS FROM NORTH INDIAN POPULATION

To clarify the sensitivity and the validity of K-ras point mutational analysis at codon 12 and 13 in North Indian patients with pancreatic diseases, and the possible correlation between the presence of the mutation and the histopathological findings. Sixty-five pancreatic ductal adenocarcinoma and 50 chronic pancreatitis patients were enrolled in this study. Codon 12 and 13 K-ras mutations were examined using the two step polymerase chain reaction (PCR) followed by single strand confirmation polymorphism (SSCP) and further confirmed by automated DNA sequencing. The positive percentage of K-ras in pancreatic carcinoma was 72.30% (47/65) which was significantly higher than that in the chronic pancreatitis 6.0% (3/50) (P A) and GGC to GGG (C>G) transitions at codon12 and codon13 respectively. These results draw attention to the critical role of K-ras gene mutation for the detection of early stage pancreatic cancer from chronic pancreatitis. K-ras gene mutation can be used as an important biomarker for early pancreatic cancer diagnosis, but it needs to be confirmed in large number of chronic pancreatitis patients.

Rapid genotyping of genetically modified laboratory animals from whole blood samples without DNA preparation

A new, rapid method is described which permits the genotyping of genetically modified animals from a microlitre volume of whole blood samples via one step polymerase chain reaction amplification. The major advantage of the presented method is the exclusion of a DNA preparation step, which significantly reduces the time expenditure and work load of the genetic testing. Pilot studies indicate, that this method is efficient and applicable also on tissue biopsies and larger amount of blood providing a rapid and reliable new technique over conventional genotyping approaches.

Development of a monoclonal antibody‐based flow‐through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black tiger shrimp Penaeus monodon

A flow-through immunoassay (FTA), an improved version of immunodot, was developed using a nitrocellulose membrane baked onto adsorbent pads enclosed in a plastic cassette to detect white spot syndrome virus (WSSV) in shrimp. Sharp purple dots developed with WSSV against the white background of the nitrocellulose membrane. The detection limits of WSSV by the FTA and immunodot were 0.312 and 1.2 μg mL(-1) crude WSSV protein, respectively. The FTA could be completed in 8-10 min compared with 90 min for immunodot. The FTA was 100 times more sensitive than 1-step polymerase chain reaction (PCR) and in between that of the 1- and 2-step PCR protocol recommended by the Office of International Epizootics (OIE). In experimental, orally infected shrimp post-larvae, WSSV was first detected 14, 16 and 18 h post-infection (hpi) by FTA, immunodot and one-step PCR, respectively. The FTA detected WSSV 2 and 4 h earlier than immunodot and one-step PCR, respectively. The FTA was more sensitive (25/27) than one-step PCR (23/27) and immunodot (23/27) for the detection of WSSV from white spot disease outbreak ponds. The reagent components of the FTA were stable giving expected results for 6 m at 4-8 °C. The FTA is available as a rapid test kit called 'RapiDot' for the early detection of WSSV under field conditions.

Two-step bacterial broad-range polymerase chain reaction analysis of heart valve tissue improves bacteriological diagnosis of infective endocarditis

Positive heart valve (HV) culture is a major Duke's criterion for the diagnosis of infective endocarditis but is poorly sensitive. Two broad-range 16S rDNA polymerase chain reaction (PCR) methods were applied to 31 HV samples: first, a real-time method, then conventional end-point PCR was applied to HV samples on which the first PCR was negative. Five specific real-time PCR procedures were also used in order to identify Bartonella spp., Tropheryma whipplei, Chlamydophila pneumoniae, Mycoplasma pneumonia, and Coxiella burnetii. A strategy combining the 2-step broad-range PCR methods improved the sensitivity of the molecular method from 38.7% to 58%. Specific PCR identified 1 T. whipplei, which was also identified by conventional end-point PCR. These results confirm that blood culture is the gold standard for the diagnosis of infective endocarditis, shows that molecular methods applied to HV can be useful when blood culture is negative, and that 2-step broad-range PCR approach seems to be more sensitive.

English

  A highly specific single step polymerase chain reaction (PCR) is described for the detection of pig (Sus domesticus) meat. A PCR assay was successfully optimized for amplification of 629 and 322-bp DNA fragment extracted from pig meat using designed species-specific primer pairs based on mitochondrial D-loop and 12Sribosomal ribonucleic acid (rRNA) gene, respectively. The optimized PCR assay was subsequently validated for its specificity with deoxyribonucleic acid (DNA) extracted from cattle, buffalo, sheep, goat and pig. PCR amplification of target DNA with pig-specific primers was repeated 15 times, with consistent results observed. The specificity of pig-specific PCR provides a valuable tool for identification of pig meat and to avoid its fraudulent substitution and adulteration.   Key words: Pig meat, adulteration, polymerase chain reaction (PCR), mitochondrialD-Loop, 12S ribosomal ribonucleic acid (rRNA) gene

English

  Brucellosis is an important disease affecting mainly sheep and goats. Diagnosis based on isolation of Brucella organisms from the suspected animals is the golden standard but has a limited sensitivity, expensive and unpractical to apply on a large scale in control campaigns. Accordingly, the indirect diagnosis of disease based on serological tests is the method of choice in the eradication programs. In this study, a single step polymerase chain reaction (PCR) was used to diagnose brucellosis using sheep whole blood and compared its sensitivity and specificity against some of the most commonly used serological techniques and modified ones. Three hundred apparently healthy ewes were randomly chosen from different governorates of Egypt. Sera were tested against Rose Bengal test (RBT), Serum Agglutination test (SAT), ELISA using both the whole Brucellaantigen (W-ELISA) and the periplasmic protein antigen (P-ELISA). Results showed that 39% of the blood samples were positive to the PCR test, Meanwhile 29.3, 27.0, 28.7 and 28.3% were positive to the previous serological tests respectively. We recommend the use of this blood PCR assay for accurate diagnosis of ovine brucellosis especially in the early stage of infection, which is difficult to achieve by the applied serological tests.   Key words: Ovine brucellosis, Blood PCR, RBT, SAT, ELISA.

Effect of processing treatments on the white spot syndrome virus DNA in farmed shrimps (Penaeus monodon)

To investigate the effect of processing treatments on the destruction of white spot syndrome virus (WSSV) DNA in WSSV-infected farmed shrimps (Penaeus monodon). The presence of WSSV was tested by single step and nested polymerase chain reaction (PCR). The primers 1s5 & 1a16 and IK1 & IK2 were used for the single step PCR and primers IK1 & IK2-IK3 & IK4 were used for the nested PCR. Various processing treatments such as icing, freezing, cooking, cooking followed by slow freezing, cooking followed by quick freezing, canning, and cold storage were employed to destroy the WSSV DNA. Of the processing treatments given, cooking followed by quick freezing was efficient in destroying WSSV DNA in WSSV-infected shrimp products. Canning, and cooking followed by slow freezing process had some destructive effect on the WSSV DNA, as WSSV DNA in such processed shrimp products was detected only by nested PCR. Icing, slow freezing, quick freezing, and cooking processes had no effect on the destruction of WSSV DNA. A gradual increase in the destruction of WSSV DNA was observed as the cold storage period increased. The results indicated that cooking followed by quick freezing process destroy the WSSV DNA. WSSV can be destroyed by cooking followed by quick freezing and this combined process can reduce the disease transmission risks from commodity shrimps to native shrimps.