Detection of Plasmodium vivax by Nested PCR and Real-Time PCR

Malaria is endemic in the Cukurova region while it is sporadic in other regions of Turkey. Therefore, the laboratory and clinical diagnosis of malaria is important for the treatment of malaria. In this study, 92 blood samples that were taken from the suspected malaria patients for routine diagnosis in a period of 10 years between 1999 and 2009 were analyzed. All of these blood samples were examined by microscopic examinations using Giemsa-stained thick blood films, nested PCR, and real-time PCR. The sensitivity-specificity and positive-negative predictive values for these diagnostic tests were then calculated. It was found that the positive predictive values of microscopic examination of thick blood films, nested PCR, and real-time PCR were 47.8%, 56.5%, and 60.9% for malaria, respectively. The real-time PCR was found to have a specificity of 75% and sensitivity of 100%, while specificity and sensitivity of nested PCR was found 81.2% and 97.7% according to the microscopic examination of thick blood films, respectively.     

Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

A nested PCR assay to detect DNA in sera for the diagnosis of deep-seated trichosporonosis

Deep-seated trichosporonosis caused by Trichosporon asahii has a high mortality rate and a very poor prognosis. New species-specific oligonucleotide primers for T. asahii were developed from a sequence analysis of rRNA genes that included the internal transcribed spacer regions. A nested PCR assay with specific primers was used to examine 11 serum samples from 7 patients, who were diagnosed with deep-seated trichosporonosis histologically at autopsy. In addition, Trichosporon cell wall polysaccharide (PS) was detected by a latex agglutination (LA) test. Of 11 samples, seven had a positive LA test, and T. asahii DNA was also detected with the nested PCR assay. Of the four samples in which PS antigen was not detected, the nested PCR of two samples was positive. Our new nested PCR assay may be used as an adjunct to conventional methods for diagnosing T. asahii infection.     

Evaluation of the use of pooled serum, pooled muscle tissue fluid (meat juice) and pooled faeces for monitoring pig herds for Salmonella

AIMS: Monitoring for Salmonella in slaughter pigs is important to enable targeted control measures to be applied on problem farms and at the abattoir. The aim of this study was to determine whether pooled serum and meat juice could be used to identify finishing pig herds with a high prevalence of infection. METHODS AND RESULTS: Samples of meat juice, serum, caecal contents, carcase swabs and pooled faeces from pig pens were taken from 20 commercial pig finishing farms and comparisons were made between the results of Salmonella culture, individual ELISA tests on serum and meat juice and pooled samples of serum and meat juice. Salmonella was isolated from samples from 19 of 20 farms. None of the ELISA tests showed a statistically significant correlation with caecal carriage of Salmonella or contamination of carcases. Serum mean optical density (O.D.) from pools of five, 10 or 20 sera showed a significant correlation with the Salmonella status of farm pen faeces. All pooled serum O.D. and sample/positive control ratio results correlated significantly with the results of the conventional individual sample ELISA. There was a statistically significant correlation between the incidence of Salmonella in farm pen pooled faeces and the prevalence of Salmonella in caeca of slaughter pigs. CONCLUSIONS: The results show a generally poor correlation between serological and bacteriological results but pooled serum or meat juice samples could be used as a cheaper substitute for serological screening of farms for Salmonella than individual samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of a cheaper test should allow the costs of Salmonella monitoring of pig farms to be reduced or allow more regular testing to enhance the designation of farm Salmonella risk status.     

A nested real-time PCR assay has an increased sensitivity suitable for detection of viruses in aerosol studies

Aims:  Influenza is commonly spread by infectious aerosols; however, detection of viruses in aerosols is not sensitive enough to confirm the characteristics of virus aerosols. The aim of this study was to develop an assay for respiratory viruses sufficiently sensitive to be used in epidemiological studies.
Method:  A two-step, nested real-time PCR assay was developed for MS2 bacteriophage, and for influenza A and B, parainfluenza 1 and human respiratory syncytial virus. Outer primer pairs were designed to nest each existing real-time PCR assay. The sensitivities of the nested real-time PCR assays were compared to those of existing real-time PCR assays. Both assays were applied in an aerosol study to compare their detection limits in air samples.
Conclusions:  The nested real-time PCR assays were found to be several logs more sensitive than the real-time PCR assays, with lower levels of virus detected at lower Ct values. The nested real-time PCR assay successfully detected MS2 in air samples, whereas the real-time assay did not.
Significance and Impact of the Study:  The sensitive assays for respiratory viruses will permit further research using air samples from naturally generated virus aerosols. This will inform current knowledge regarding the risks associated with the spread of viruses through aerosol transmission.     

PCR diagnosis of Entamoeba histolytica cysts in stool samples

Amebiasis is a protozoan disease caused by Entamoeba histolytica and a potential health threat in areas where sanitation and hygiene are inappropriate. Highly sensitive PCR methods for detection of E. histolytica in clinical and environmental samples are extremely useful to control amebiasis and to promote public health. The present study compared several primer sets for small subunit (SSU) rDNA and histone genes of E. histolytica cysts. A 246 bp of the SSU rDNA gene of pure cysts contained in phosphate-buffered saline (PBS) and in stool samples was successfully amplified by nested PCR, using the 1,147-246 bp primer set, of the primary PCR products which were pre-amplified using the 1,147 bp primer as the template. The detection limit of the nested PCR using the 1,147-246 primer set was 10 cysts in both groups (PBS and stool samples). The PCR to detect histone gene showed negative results. We propose that the nested PCR technique to detect SSU rDNA can be used as a highly sensitive genetic method to detect E. histolytica cysts in stool samples.     

Evaluation of nested PCR in detection of Helicobacter pylori targeting a highly conserved gene: HSP60

Objective: To comparatively evaluate a new nested set of primers designed for the detection of Helicobacter pylori targeting a highly conserved heat shock protein gene (Hsp60). Methods: A total of 60 subjects having peptic ulcer diseases were tested for the detection of H. pylori using rapid urease test (RUT), histology, culture, and polymerase chain reaction (PCR) in their antral biopsy specimens. A newly designed Hsp60 gene‐based primer set was evaluated against commonly used PCR primers for detection of H. pylori. Results: Forty‐six of the 60 study subjects were found positive for culture isolation and all the 46 culture‐positive specimens were also positive with Hsp60 gene PCR. Of the 46 culture‐positive specimens, 44 were positive for 16S rRNA gene, ureC gene, RUT, and histology whereas only 29 were positive with ureA gene PCR. Of the 14 culture‐negative subjects, 10 were positive with 16S rRNA gene, 4 were positive with ureC (glmM) gene PCR, and 2 were positive with RUT and 1 was positive on histology. Conclusion: This study shows that nested amplification targeting Hsp60 gene is the most sensitive and specific with LR⁺ and LR^? values of ∝ and 0, respectively, when compared with the other three PCR methods. Also, HSP60 gene‐specific nested protocol was the most appropriate for detection of H. pylori in clinical specimens. This is particularly valuable because it can be used as a noninvasive method for detecting H. pylori infection in young children and also, in follow‐up studies with peptic ulcer patients, on samples like feces and saliva.     

Accuracy of a routine real-time PCR assay for the diagnosis of Pneumocystis jirovecii pneumonia

Pneumocystis jirovecii is a common cause of life-threatening pneumonia among immunocompromised patients. Using 400 fresh bronchoalveolar lavage samples, we compared prospectively routine direct immunofluorescence assay (DFA) and a real-time PCR assay, performed on a LightCycler system, for the detection of P. jirovecii. Among the 66 PCR positive samples, 31 were positive by DFA. No patient was found as having the pattern "PCR--ve/DFA+ve". The semi-quantification of the P. jirovecii DNA was represented by the cycle threshold (Ct). Using DFA as the gold standard, the sensitivity of the PCR was 100% for Ct>/=28 and the specificity was 100% for Ct<22. Between these two points, the results could be discrepant. The patients of the "22/=28" group, and presented less frequently with HIV-infection and elevated lactate dehydrogenase (LDH) assay than in the "Ct<22" group. A negative PCR allowed us to exclude the P. jirovecii pneumonia. The real-time PCR assay seems to be an accurate diagnosis method and could replace the DFA. The semi-quantitative results should be helpful to distinguish colonized, subclinically infected and P. jirovecii pneumonia patients.     

Trichomonas vaginalis detection using real-time TaqMan PCR

1978 women and 93 men, all suspected of having a Trichomonas vaginalis infection, were tested for the presence of T. vaginalis by real-time PCR using the T. vaginalis-specific 2-kb repeated sequence, and by direct microscopy and culture. 40 samples were positive by T. vaginalis real-time PCR and 27 were positive by wet mount microscopy, either direct or after culture. All samples positive by direct microscopy of culture were also positive by real-time PCR. Of the 13 samples which were real-time PCR positive but negative by direct microscopy and culture 11 were confirmed by another T. vaginalis real-time PCR based on the beta tubulin gene. Only 2 samples (0.1%) showed inhibition in the PCR. The prevalence of T. vaginalis infection in the female patients was 1.8%. The sensitivity, specificity, positive and negative predictive values of the real-time PCR were 100%, 99.9%, 95% and 100%, respectively. The same test characteristics for the combined conventional T. vaginalis detection methods (microscopy+culture) were 71%, 100%, 100% and 99%, respectively. Therefore, real-time PCR is the method of choice for the diagnosis of T. vaginalis infection.     

Simultaneous detection of waterborne viruses by multiplex real-time PCR

Norovirus, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus are all common causes of gastroenteritis. Conventional diagnoses of these causative agents are based on antigen detection and electron microscopy. To improve the diagnostic potential for viral gastroenteritis, internally controlled multiplex real-time polymerase chain reaction (PCR) methods have been recently developed. In this study, individual real-time PCRs were developed and optimized for specific detections of Norovirus genogroup I, Norovirus genogroup II, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus group B1. Subsequently, individual PCRs were combined with multiplex PCR reactions. In general, multiplex real-time PCR assays showed comparable sensitivities and specificities with individual assays. A retrospective clinical evaluation showed increased pathogen detection in 29% of samples using conventional PCR methods. Prospective clinical evaluations were detected in 123 of the 227 (54%) total samples used in the multiplex real-time PCR analysis. The Norovirus genogroup II was found most frequently (23%), followed by Rotavirus (20%), the Hepatitis A virus (4.5%), Coxsackievirus (3.5%), and Norovirus genogroup I (2.6%). Internally controlled multiplex real-time PCR assays for the simultaneous detection of Rotavirus, Coxsackievirus group B, the Hepatitis A virus, and Norovirus genogroups I and II showed significant improvement in the diagnosis of viral gastroenteritis.     

Rapid and effective detection of anthrax spores in soil by PCR

AIMS: To detect Bacillus anthracis DNA from soil using rapid and simple procedures. METHODS AND RESULTS: Various amounts of B. anthracis Pasteur II spores were added artificially to 1 g of soil, which was then washed with ethanol and sterile water. Enrichment of the samples in trypticase soy broth was performed twice. A DNA template was prepared from the second enrichment culture using a FastPrep instrument. The template was then used for nested and real-time polymerase chain reaction (PCR) with B. anthracis-specific primers, to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. CONCLUSIONS: One cell of B. anthracis in 1 g of soil could be detected by nested and real-time PCR. The usefulness of the PCR method using field samples was also confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that this could be a useful method for detecting anthrax-spore contaminated soil with high sensitivity. Its application could have great impact on the progress of epidemiological surveillance.     

Still Searching for a Suitable Molecular Test to Detect Hidden Plasmodium Infection: A Proposal for Blood Donor Screening in Brazil

Background

Efforts have been made to establish sensitive diagnostic tools for malaria screening in blood banks in order to detect malaria asymptomatic carriers. Microscopy, the malaria reference test in Brazil, is time consuming and its sensitivity depends on microscopist experience. Although molecular tools are available, some aspects need to be considered for large-scale screening: accuracy and robustness for detecting low parasitemia, affordability for application to large number of samples and flexibility to perform on individual or pooled samples.

Methodology

In this retrospective study, we evaluated four molecular assays for detection of malaria parasites in a set of 56 samples previously evaluated by expert microscopy. In addition, we evaluated the effect of pooling samples on the sensitivity and specificity of the molecular assays. A well-characterized cultured sample with 1 parasite/μL was included in all the tests evaluated. DNA was extracted with QIAamp DNA Blood Mini Kit and eluted in 50 μL to concentrate the DNA. Pools were assembled with 10 samples each. Molecular protocols targeting 18S rRNA, included one qPCR genus specific (Lima-genus), one duplex qPCR genus/Pf (PET-genus, PET-Pf) and one duplex qPCR specie-specific (Rougemont: Roug-Pf/Pv and Roug-Pm/Po). Additionally a nested PCR protocol specie-specific was used (Snou-Pf, Snou-Pv, Snou-Pm and Snou-Po).

Results

The limit of detection was 3.5 p/μL and 0.35p/μl for the PET-genus and Lima-genus assays, respectively. Considering the positive (n = 13) and negative (n = 39) unpooled individual samples according to microscopy, the sensitivity of the two genus qPCR assays was 76.9% (Lima-genus) and 72.7% (PET-genus). The Lima-genus and PET-genus showed both sensitivity of 86.7% in the pooled samples. The genus protocols yielded similar results (Kappa value of 1.000) in both individual and pooled samples.

Conclusions

Efforts should be made to improve performance of molecular tests to enable the detection of low-density parasitemia if these tests are to be utilized for blood transfusion screening.     

Adding a selective enrichment step to the iQ-Check real-time PCR improves the detection of Salmonella in naturally contaminated retail turkey meat products

AIMS: The aim of this study was to compare the real-time iQ-Check Salmonella kit (Bio-Rad) with the immunocapture assay RapidCheck Salmonella method, and a conventional culture method (FSIS, USDA) in detecting Salmonella in naturally contaminated turkey meat products. This study was also designed to determine if a selective enrichment step might improve the real-time detection of Salmonella. METHODS AND RESULTS: Using the culture method, Salmonella was recovered from 49 out of 99 retail turkey meat samples collected. RapidCheck failed to detect 11 Salmonella samples that were positive by the culture method. The iQ-Check real-time PCR also failed to detect three samples that were positive by the culture method. However, when carried out after a selective enrichment step, the iQ-Check real-time PCR detected all 49 Salmonella samples recovered by the culture method. The iQ-Check real-time PCR detected the presence of Salmonella in some samples that were not recovered by the culture method. CONCLUSIONS: Adding a selective enrichment step to the iQ-Check real-time PCR improves the detection of Salmonella in naturally contaminated turkey meat samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The iQ-Check Salmonella real-time PCR can be used as a rapid method to monitor Salmonella in turkey meat, together with conventional culture methodology.     

Real-time PCR versus conventional PCR for malaria parasite detection in low-grade parasitemia

We have optimized a faster and cheaper real-time PCR and developed a conventional genus specific PCR based on 18S rRNA gene to detect malaria parasites in low-grade parasitemias. Additionally, we compared these PCRs to the OptiMAL-IT test. Since there is no consensus on choice of standard quantitative curve in real-time assays, we decided to investigate the performance of parasite DNA from three different sources: "genome", amplicon and plasmid. The amplicon curve showed the best efficiency in quantifying parasites. Both PCR assays detected 100% of the clinical samples tested; the sensitivity threshold was 0.5 parasite/mul and no PCR positive reaction occurred when malaria parasites were not present. Conversely, if OptiMAL-IT were employed for malaria diagnosis, 30% of false-negative results could be expected. We conclude that PCR assays have potential for detecting malaria parasites in asymptomatic infections, in evaluation of malaria vaccine molecule candidates, for screening blood donors, especially in endemic areas, or even in monitoring malaria therapy.     

Widespread use of real-time PCR for rickettsial diagnosis

We report 2?years of experience with rickettsial molecular diagnosis using real-time PCR at the French National Reference Center. All Rickettsia genomes available were compared to discover specific sequences to design new sets of primers and probes. The specificity was verified in silico and against a panel of 30 rickettsial species. Sensitivity was determined using 10-fold serial dilutions. Finally, primers and probes that were both specific and sensitive were routinely used for the diagnosis of rickettsial infections from clinical specimens. We retained sets of primers and probes to detect spotted fever group Rickettsia, typhus group Rickettsia,Rickettsia conorii,Rickettsia slovaca,Rickettsia africae and Rickettsia australis; 643 clinical samples were screened for the presence of Rickettsia DNA. Overall, 45 positive samples were detected, including 15 Rickettsia africae, nine R.?conorii, five Rickettsia sibirica mongolitimonae, four R.?slovaca, two R.?australis, four Rickettsia massiliae, one Rickettsia honei, one Rickettsia typhi and eight Rickettsia sp. Positive samples were detected mainly from cutaneous biopsies and swabs (31/45). Widespread use of real-time PCR is inexpensive and reduces delay in the diagnosis of rickettsial infections. These real-time PCR assays could be implemented easily in laboratories that have molecular facilities and may be added to existing molecular tools as a point-of-care strategy.     

Evaluation of bioelectronics sensor compared to other diagnostic test in diagnosis of Johne's disease in goats

A bioelectronics sensor has been developed and it is evaluated for the diagnosis of paratuberculosis in goats. Initially hematite nanoparticles were prepared and using this nanoparticles as core, electrically active polyaniline coated magnetic (EAPM) nanoparticles are synthesized from aniline monomer (made electrically active by acid doping). These EAPM nanoparticles were fabricated with rabbit anti-goat IgG for the detection of goat antibodies on the capture pad. The protoplasmic antigen of Mycobacterium avium subspecies paratuberculosis (MAP) immobilized onto the capture pad will detect the antibody against MAP in the goat sera samples. This bound goat antibody will be detected by the anti-goat IgG previously bound to EAPM. Upon detection the EAPM nanoparticles bridges an electric circuit between the silver electrodes, flanking the capture membrane. The electrical conductance, caused by EAPM, was measured as direct charge transfer between the electrodes. Testing of the biosensor with known Johne's disease (JD) positive and negative serum samples gave significant difference in the electrical conductance value. Further the efficacy of this biosensor was compared with other serological tests like agar gel immunodiffusion (AGID) and absorbed ELISA using field sera. Out of 265 goat sera tested, positive results recorded were; AGID 36 (13.59%), bioelectronics sensor 49 (19.14%), and absorbed ELISA 51 (19.25%). This biosensor was also compared in live animals using intradermal Johnin test and nested PCR (detecting mycobacterial DNA in feces) in 65 animals. Of which, positive results recorded in animals were; Johnin test 21 (32%), biosensor 26 (40%) and fecal PCR detected mycobacterial DNA in 28 (43%) animals. Though the nanobioelectronics sensor was slightly less sensitive (not statistically significant) compared to absorbed ELISA and fecal nested PCR for mycobacterial DNA but it was simple to perform in field conditions and requires less time. The speed of detection and the equipment involved would support its application toward the various point-of-care opportunities aimed at control and management of Johne's disease in goats.     

Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia

In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit < 10 yeast cells. Of the 18 blood samples tested daily over a period of 8 days following Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.     

Culture and PCR analysis of joint fluid in the diagnosis of prosthetic joint infection

This prospective study compared PCR and culture techniques in the diagnosis of prosthetic joint infection (PJI). We obtained joint fluid samples (JFS; n=115) from patients who had failed total joint arthroplasty between January 2003 and June 2005; 49 were positive for PJI according to established strict criteria. JFS were analyzed by PCR (n=35; control n=66) or culture (n=46, control n=48). PCR was positive in 71% of PJI cases, resulting in sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and likelihood ratio for positive results as follows: 0.71; 0.97; 0.88; 0.93; 0.87 and 23.6, respectively. Culture was positive in 44% of PJI samples. Corresponding statistics were 0.44; 0.94; 0.69; 0.87; 0.63 and 7.0, respectively. Significantly higher sensitivity, accuracy and negative predictive values were calculated for PCR versus culture, and there was 83% concordance between the results of intraoperative culture and PCR detection of causative bacteria. Therefore, we conclude that PCR analysis of synovial fluid increases the utility of pre-operative aspiration for patients who require revision total joint surgery.     

运用套式PCR检测传染性喉气管炎病毒核酸

选择鸡传染性喉气管炎病毒保守TK基因的蛋白编码区域,设计并合成了一对外引物和一对内引物,建立并优化了检测鸡传染性喉气管炎病毒DNA的套式PCR法。通过检测ILTV感染的鸡胚绒毛尿囊膜,实验室病料和临床病料,结果表明,套式PCR法能检测出ILTV感染后的非免疫鸡胚和SPF鸡绒毛膜研磨液中的被稀释了10^5倍的病毒（约1fg的ILTV DNA）,攻毒后第10天还能从非免疫鸡和SPF鸡气管拭子中检出ILTV,第10天非免疫鸡气管拭子中ILTV的最大检出率为7／10,第10天SPF鸡气管拭子中ILTV的最大检出率为8／10。对非免疫鸡和SPF鸡的气管拭中ILTV最佳检出时间均在攻毒后第5天。对临床样品中的ILTV的最大检出率为7／7。经过核酸杂交验证,套式PCR法具有很高的特异性和敏感性,为从分子水平探讨ILTV的发病机理、临床早期快速诊断提供了新的研究手段。     

Nested real-time PCR for hepatitis A detection

Aims:  The hepatitis A virus (HAV) is one of the most important human foodborne pathogens causing a number of worldwide outbreaks each year. The detection of HAV in food samples remains a complex issue, because commonly used detection tools, such as conventional or even real-time PCR assays, are often unable to detect HAV with sufficient sensitivity. The aims of this study were to develop highly sensitive and specific nested real-time PCR (NRT-PCR)-based method for HAV detection in food and to compare it with currently available methods.
Methods and Results:  By combining conventional PCR, nested PCR and real-time PCR techniques, we have developed a specific NRT-PCR assay for the detection of HAV. The procedure involves two consecutive PCRs, the first of which is performed as a conventional RT-PCR using primers specific for HAV 5' noncoding region. The second reaction involves a real-time PCR using a nested primer pair specific for the first PCR product and a TaqMan probe.
Conclusions:  We have developed a novel NRT-PCR method capable of detecting as little as 0·2 PFU of HAV, which is significantly more sensitive than any other PCR technique tested in our system.
Significance and Impact of the Study:  NRT-PCR provides a potentially useful method for detecting HAV at extremely low levels, as frequently found in food samples, and can be potentially adopted as a regulatory method to ensure food safety.