Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus,hepatitis B virus,and human immunodeficiency virus 1

BACKGROUND Hepatitis C virus(HCV),hepatitis B virus(HBV),and human immunodeficiency virus 1(HIV-1)are the most epidemic blood-borne viruses,posing threats to human health and causing economic losses to nations for combating the infection transmission.The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive,but they are more accurate than sero-logical testing.AIM To develop a rapid,cost-effective,and accurate diagnostic multiplex polymerase chain reaction(PCR)assay for simultaneous detection of HCV,HBV,and HIV-1.METHODS The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electro-phoretic molecular weight inside each viral genome.Therefore,this diagnostic method will be appropriate for application in both conventional(combined with electrophoresis)and real-time PCR facilities.Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus.Then,Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay.RESULTS The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay.The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis.Compared to related published assays,the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays.CONCLUSION This study provides a simple,cost-effective,and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses;this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

Locked nucleic acid real-time polymerase chain reaction method identifying two polymorphisms of hepatitis B virus genotype C2 infections,rt269L and rt269I

BACKGROUND The presence of two distinct hepatitis B virus(HBV)Pol RT polymorphisms,rt269L and rt269I,could contribute to the unique clinical or virological phenotype of HBV genotype C2.Therefore,a simple and sensitive method capable of identifying both types in chronic hepatitis B(CHB)patients infected with genotype C2 should be developed.AIM To develop a novel simple and sensitive locked nucleic acid(LNA)-real timepolymerase chain reaction(RT-PCR)method capable of identifying two rt269 types in CHB genotype C2 patients.METHODS We designed proper primer and probe sets for LNA-RT-PCR for the separation of rt269 types.Using synthesized DNAs of the wild type and variant forms,melting temperature analysis,detection sensitivity,and endpoint genotyping for LNA-RT-PCR were performed.The developed LNA-RT-PCR method was applied to a total of 94 CHB patients of genotype C2 for the identification of two rt269 polymorphisms,and these results were compared with those obtained by a direct sequencing protocol.RESULTS The LNA-RT-PCR method could identify two rt269L and rt269I polymorphisms of three genotypes,two rt269L types[‘L1’(WT)and‘L2’]and one rt269I type(‘I’)in single(63 samples,72.4%)or mixed forms(24 samples,27.6%)in 87(92.6%sensitivity)of 94 samples from Korean CHB patients.When the results were compared with those obtained by the direct sequencing protocol,the LNA-RT-PCR method showed the same results in all but one of 87 positive detected samples(98.9%specificity).CONCLUSION The newly developed LNA-RT-PCR method could identify two rt269 polymorphisms,rt269L and rt269I,in CHB patients with genotype C2 infections.This method could be effectively used for the understanding of disease progression in genotype C2 endemic areas.

Small Amplicons Mutation Library for Vaccine Screening by Error-Prone Polymerase Chain Reaction

Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.

Confusing finding of quantitative fluorescent polymerase chain reaction analysis in invasive prenatal genetic diagnosis:A case report

BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing.

Deoxyribonucleic Acid-Polymerase Chain Reaction Status of HIV Exposed Infants in a Sub Regional Prevention of Mother-to-Child Transmission of HIV Programme during the Period 2009-2020

Introduction: Transitioning to more efficacious Antiretrovirals for HIV infected pregnant women and infant prophylaxis has reduced Mother to child transmission of HIV significantly. This study aimed to determine HIV infection status in HIVexposed infants who had their first DNA polymerase chain reaction test in our molecular Laboratory. Subjects, Materials and Methods: Dried Blood Spots for HIV DNA results from 5 states between 2009 and 2020 were analyzed in the PCR laboratory of the Federal Teaching Hospital, Gombe. Results: Nine thousand eight hundred and twenty-three Human Immunodeficiency Virus Deoxyribonucleic acid polymerase Chain Reaction results were analysed;4937 (50.2%) were males. During the study period, there was an overall declining trend in the mother-to-child transmission rate from 3.8% in 2009 to 1.0% in 2020. 6120 (62.3%) of HIV + mothers received Highly active antiretroviral therapy HAART before pregnancy. 7845 (76.2%) of the infants received Nevirapine prophylaxis. Dried blood spot samples were collected from 4077 (41.5%) at 6 - 8 weeks. 8438 (85.9%) received cotrimoxazole. 9469 (96.4%) were ever breastfed. Of the 9823 HIV DNA PCR results, 255 (2.6%) were positive while 69/4077 (1.7%) and 109/2662 (4.1%) were positive for HIV DNA at 6 - 8 weeks and > 12 weeks respectively. (p = 0.001). 86/747 (11.5%) of infants whose HIV-positive mothers received no ARVS were HIV DNA positive. (p = 0.001). 106/884 (12.0%) of infants who had no Antiretroviral prophylaxis had positive HIV DNA results;7/413 (1.7%) with Zidovudine/Nevirapine prophylaxis had positive results. (p = 0.001). 246/9469 (2.6%) of infants that were ever breastfed were positive for HIV DNA;11/354 (3.0%) that never breastfed had positive HIV DNA. Conclusion: Lack of maternal/infant ARVs and prolonged breastfeeding increased the risk of infant HIV infection.

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing *

AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.

Detection of Neisseria Gonorrhoeae from Urine with Ligase Chain Reaction

Objective: To evaluate the value of ligase chainreaction(LCR) in the diagnosis of diplococcusgonorrhoeae in urine. Methods: LCR detection of the urine for Neisseriagonorrhoeae and bacteria culture of discharge was per-formed simultaneously to 276 patients with urethritisor cervicitis seeking treatment in sex transmitted dis-eases (STDs) outpatient clinic. For specimens withdiscordant results, polymerase chain reaction wasconducted. The purpose was to detect the respectivesensitivity and specificity of bacteria culture and LCR. Results: 24 of 276(8.7%) patients had positive LCRresults and 21 of 276(7.6%) were positive for culture.5 specimens had discordant results from LCR andbacteria culture. The sensitivity and specificity of LCRin the diagnosis of gonorrhoeae were 92.3% and100% respectively. Conclusion: This study showed that LCR had ahigher sensitivity and specificity for the diagnosis ofgonorrhoeae from urine.

Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction

AIM： To examine the sensitivity and accuracy of real-time polymerase chain reaction （PCR） for the quantification of hepatitis B virus （HBV） DNA in semen. METHODS： Hepatitis B viral DNA was isolated from HBV carriers＇ semen and sera using phenol extraction method and QIAamp DNA blood mini kit （Qiagen, Germany）. HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR （quantitative polymerase chain reaction （qPCR））. The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. RESULTS： Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×10^7 and 1.67×10^7 copies of HBV DNA per mL in two HBV infected patients＇ sera, while 2.14×10^5 and 3.02×10^5 copies of HBV DNA per mL in the semen. CONCLUSION： Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.

Rapid detection of sepsis complicating acute necrotizing pancreatitis using polymerase chain reaction

INTRODUCTIONAcute narcotizing pancreatitis usually takes a severe clinical course and is associated with multiple organ dysfunction .With the further understanding of pathophysiological events of acute pancreatisis and the therapeutic measuses taken by the clinicians ,the patients can pass through the critical carry stages ,and then the septic complication caused by rtanslocated bacteria, mostly gram-negative microbes from the intestines ensues[1].

Detection of PERV by polymerase chain reaction and its safety in bioartificial liver support system

AIM： To establish a method detecting porcine endogenous retrovirus （PERV） in China experimental minipigs and to evaluate the safety of PERV in three individuals treated with bioartificial liver support systems based on porcine hepatocytes. METHODS： Porcine hepatocytes were isolated with two-stage perfusion method, then cultured in the bioreactor, which is separated by a semipermeable membrane （0.2μm） from the lumen through which the patients＇ blood plasma was circulated. After posthemoperfusion, patients＇ blood was obtained for screening. Additionally, samples of medium collected from both intraluminal and extraluminal compartments of the laboratory bioreactor and culture supernate in vitro was analyzed. The presence of viral sequences was estimated by polymerase chain reaction （PCR） and reverse transcriptase-polymerase chain reaction （RTPCR）. Finally, the infection of virus in the supernate of common culture was ascertained by exposure to the fetal liver cells. RESULTS： PERV-specific gag sequences were found in the porcine hepatocytes using RT-PCR. and were detected in all samples from the intraluminal, extraluminal samples and culture supernate. However, culture supernatant from primary porcine hepatocytes （cleared of cellular debris） failed to infect human fetal liver cells. Finally, RT-PCR detected no PERV infection was found in the blood samples obtained from three patients at various times post-hemoperfusion. CONCLUSION： The assays used are specific and sensitive, identified by second PCR. PERVs could be released from hepatocytes cultured in bioreactor without the stimulation of mitogen and could not be prevented by the hollow fiber semipermeable membrane, indicating the existence of PERV safety in extracorporeal bioartificial liver support system （EBLSS）.

Using the polymerase chain reaction coupled with denaturing gradient gel electrophoresis to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted acute severe pancreatitis

AIM: To investigate the use of PCR and DGGE to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted severe AP.METHODS: Patients with biochemical and clinical evidence of acute pancreatitis and an APACHE Ⅱ score ≥8 were enrolled. PCR and DGGE were employed to detect bacterial translocation in blood samples collected on d1,3, and 8 after the admission. Standard microbial blood cultures were taken when there was clinical evidence of sepsis or when felt to be clinically indicated by the supervising team.RESULTS: Six patients were included. Of all the patients investigated, only one developed septic complications;the others had uneventful illness. Bacteria were detected using PCR in 4 of the 17 collected blood samples. The patient with sepsis was PCR-positive in two samples (taken on d 1 and 3), despite three negative blood cultures. Using DGGE and specific primers, the bacteria in all blood specimens which tested positive for the presence of bacterial DNA were identified as E coli.CONCLUSION: Our study confirmed thatunlike traditional microbiological techniques, PCR can detect the presence of bacteria in the blood of patients with severe AP. Therefore, this latter method in conjunction with DGGE is potentially an extremely useful tool in predicting septic morbidity and evaluating patients with the disease. Further research using increased numbers of patients, in particular those patients with necrosis and sepsis, is required to assess the reliability of PCR and DGGE in the rapid diagnosis of infection in AP.

Gastrointestinal tract distribution of Salmonella enteritidis in orally in fected mice with a species-specific fluorescent quantitative polymerase chain reaction

AIM: To identify and understand the regular distribution pattern and primary penetration site for Salmonella enteritidis (S. enteritidis) in the gastrointestinal tract. METHODS: Based on the species-specific DNA sequence of S. enteritidis from GenBank, a species-specific real- time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) was developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the gastrointestinal tract, including duodenum, jejunum, ileum, cecum, colon, rectum, esophagus and stomach, from mice after oral infection. RESULTS: S. enteritidis was consistently detected in all segments of the gastrointestinal tract. The jejunum and ileum were positive at 8 h post inoculation, and the final organ to show a positive result was the stomach at 18 h post inoculation. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h post inoculation, with the jejunum, ileum and cecum containing high concentrations of S. enteritidis, whereas the duodenum, colon, rectum, stomach and esophagus had low concentrations. S. enteritidis began to decrease and vanished at 2 d post inoculation, but it was still present up to 5 d post inoculation in the jejunum, ileum andcecum, without causing apparent symptoms. By 5 d post inoculation, the cecum had significantly higher numbers of S. enteritidis than any of the other areas (P < 0.01), and this appeared to reflect its function as a repository for S. enteritidis. CONCLUSION: The results provided significant data for clarifying the pathogenic mechanism of S. enteritidis in the gastrointestinal tract, and showed that the jejunum, ileum and cecum are the primary sites of invasion in normal mice after oral infection. This study will help to further understanding of the mechanisms of action of S. enteritidis.

Quantitative studies of the regular distribution pattern for Salmonella enteritidis in the internal organs of mice after oral challenge by a specific real-time polymerase chain reaction

AIM： To identify and understand the regular distribution pattern for Salmonella enteritidis （S. enteritidis） in the internal organs of mice after an oral challenge over a 3 wk period. METHODS： Assays based on the serovar-specific DNA sequence of S. enteritidis from GenBank, and a serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction （FQ-PCR） were developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the blood and the internal organs, including heart, liver, spleen, kidney, pancreas, and gallbladder, from mice after oral challenge at different time points respectively.RESULTS： The results showed that the spleen was positive at 12 h post inoculation （PI）, and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, the pancreas was positive at 20 h PI, and the final organs to show positive results were the kidney and gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h PI, with the liver and spleen containing high concentrations of S. enteritidis, whereas the blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 12 d PI in the gallbladder, 2 wk for the liver, and 3 wk for the spleen without causing apparent symptoms.CONCLUSION： The results provided significant data for understanding the life cycle of S. enteritidis in the internal organs, and showed that the liver and spleen may be the primary sites for setting itself up as a commensa over a long time after oral challenge. Interestingly, it may be the first time reported that the gallbladder is a site of carriage for S. enteritidis over a 12 d period. This study will help to understand the mechanisms of action of S. enteriCdis infection in vivo.

Optimization of competitively differentiated poiymerase chain reaction in detection of HBV basal core promoter mutation

AIM:To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation. METHODS: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control. To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CD-PCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection. RESULTS: The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to 10μmol/L and mutual primer to about 100μmol/L Optimized CD-PCR could detect both mutant and wild strain independent of the amount of templates and the number of PCR cycles. Its detection limit was 103 copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg. CONCLUSION: Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles.

Detection of circulating hepatocellular carcinoma cells in peripheral venous blood by reverse transcription-polymerase chain reaction

Objective: To detect circulating hepatocellular carcino-ma by demonstrating hepatocellular carcinoma cells orhepatocyte-associated mRNA in the nuclear cell com-ponent of peripheral blood (PBL).Methods: Peripheral blood (5 ml) samples were ob-tained from 93 patients with hepatocellular carcinoma(HCC) and from 33 control subjects (9 with liver cir-rhosis after hepatitis B,14 with chronic hepatitis B,10with normal liver function). To identify HCC cells inperipheral blood, liver-specific human alpha-fetopro-tein (AFP) mRNA was amplified from total RNA ex-tracted from whole blood by reverse transcription-polymerase chain reaction.Results: AFPmRNA was detected in 50 blood samplesfrom the HCC patients (50/93, 53.8%). In contrast,there were no clinical control patients whose samplesshowed detectable AFPmRNA in PBL. The presence ofAFPmRNA in blood seemed to be correlated with thestage (by TNM classification) of HCC, the serum AFPvalue, and the presence of intrahepatic metastasis,portal vein thrombosis, tumor diameter and/or distantmetastasis. In addition, AFPmRNA was detected in theblood of 21 patients with metastasis at extrahepaticorgans (100%) in contrast to 29 (40.3%)of 72 pa-tients without metastasis.Conclusion: The presence of AFPmRNA in peripheralblood may be an indicator of malignant hepatocytes,which might predict hematogenous spreading metasta-sis of tumor cells in patients with HCC.

Human papillomavirus 16 physical status detection in preinvasive and invasive cervical carcinoma by multiplex real-time polymerase chain reaction

Objective： To explore an ideal approach for detecting the physical status of HPV-16 in clinic use and to investigate the integrated HPV-16 in CINs and cervical cancer. Methods： Multiplex real-time PCR method was established to quantify the copy numbers of E2 and E6 genes （E2/E6） for analysis of the physical status of HPV-16 DNA and this assay was compared to Southern blot analysis. HPV-16-containing paraffin-embedded tissues including 49 CINs and 51 cervical squamous cancers were detected using the method. Results： （1） The cutoff ratio of E2/E6 to distinguish pure episomal from mixed HPV-16, was 0.81 in the multiplex real-time PCR; （2） The agreement rate between multiplex real-time PCR and Southern blot was 81.5% （the Kappa statistic was 0.844, P〈0.001）; （3） HPV-16 DNA existed in an episomal form in 57.1% and mixed form in 42.9% of CIN I lesions; The concomitant form of HPV-16 （〉70%） constituted the majodty in CIN Ⅱ and CIN Ⅲ; HPV-16 DNA mostly integrated into the host chromosome （s） in squamous cervical cancers （68.6%）; （4） The incidence of HPV-16 integration was increased with the degree of cervical lesions; （5） The frequency of pure integrated HPV-16 in stage Ⅱ＋Ⅲ （88%） was significantly higher than that in stage Ⅰ （33.3%）. Conclusion： （1） Mutiplex real-time PCR provides a rapid, sensitive and reliable method for clinic detection of the physical state of HPV-16 DNA; （2） The integration of the HPV-16 DNA is a very eady and important event in the progression from preinvasive to invasive cervical cancer; （3） The pure integrated status of HPV-16 in cervical cancer may be associated with poor prognosis of cervical cancer, but further study will be needed to prove its prognostic significance.

Mucosal polymerase chain reaction for diagnosing Helicobacter pylori infection in patients with bleeding peptic ulcers

AIM: Helicobacter pylori (H pylon) has been linked to chronic gastritis, peptic ulcers, gastric cancer and MALT-lymphoma. Conventional invasive tests are less sensitive than non-invasive tests in diagnosing H pylori infection in patients with bleeding peptic ulcers. Polymerase chain reaction is a sensitive and accurate method for diagnosing H pylori infection. The aim of this study was to evaluate the diagnostic role of mucosai polymerase chain reaction for H pylori infection in patients with bleeding peptic ulcers. METHODS: In patients with bleeding, non-bleeding peptic ulcers and chronic gastritis, we checked rapid urease test, histology, bacterial culture and mucosai polymerase chain reaction fordetecting H pylori infection. Positive H pylori infection was defined as positive culture or both a positive histology and a positive rapid urease test. For mucosai polymerase chain reaction of H pylori, we checked vacA (s1a, s1b, s1c, s2, m1, m1T, m2), iceA1, iceA2. and cag A. RESULTS: Between October 2000 and April 2002,88 patients with bleeding peptic ulcers (males/females: 60/28, gastric ulcers/duodenal ulcers: 55/33), 81 patients with non-bleeding peptic ulcers (males/females: 54/27, gastric ulcers/duodenal ulcers: 45/36) and 37 patients with chronic gastritis (males/ females: 24/13) were enrolled in this study. In patients with bleeding peptic ulcers, non-bleeding peptic ulcers and chronic gastritis, 45 patients (51%), 71 patients (88%) and 20 patients (54%) respectively were found to have positive H pylori infection (P<0.001). In patients with bleeding peptic ulcers, non-bleeding peptic ulcers and chronic gastritis, polymerase chain reaction for H pylori infection was positive in 54 patients (61%), 70 patients (86%) and 20 patients (54%) respectively (P<0.001). The sensitivity, positive predictive value and diagnostic accuracy of mucosai polymerase reaction for H pylori infection were significantly lower in patients with bleeding peptic ulcers (84%, 79% and 81%) than in patients with non-bleeding peptic ulcers (99%, 99% and 98%) (P<0.001, P<0.01 and P<0.001 respectively). The sensitivity, negative predictive value and diagnostic accuracy of mucosal polymerase reaction for H py/ori were significantly lower in patients with bleeding peptic ulcers (84%, 83% and 81%) than in patients with chronic gastritis (100%, 100% and 100%) (P= 0.02, P= 0.02 and P=0.001). CONCLUSION: Mucosal polymerase chain reaction for detecting H pylori infection is not reliable in patients with bleeding peptic ulcers.

Polymerase chain reaction: A sensitive method for detecting Helicobacter pyloriinfection in bleeding peptic ulcers

AIM: To assess the sensitivity and specificity of polymerase chain reaction (PCR) in detecting Helicobacter pylori(H pylon) infection in patients with bleeding peptic ulcers, and to compare its diagnostic efficacy with other invasive and non-invasive tests. METHODS: From April to September 2002, H pylori status in 60 patients who consecutively presented with gastroduodenal ulcer bleeding was examined by rapid urease tests (RUT), histology, culture, PCR, serology and urea breath tests (UBT). RESULTS: The sensitivity of PCR was significantly higher than that of RUT, histology and culture (91% vs 66%, 43% and 37%, respectively; P = 0.01, <0.001, <0.001, respectively), but similar to that of serology (94%) and UBT (94%). Additionally, PCR exhibited a greater specificity than serology (100% vs 65%, P<0.01). However, the specificity of PCR did not differ from that of other tests. Further analysis revealed significant differences in the sensitivities of RUT, culture, histology and PCR between the patients with and those without blood in the stomach (P<0.01, P= 0.09, P<0.05, and P<0.05, respectively). CONCLUSION: PCR is the most accurate method among the biopsy-based tests to detect H pylori infection in patients with bleeding peptic ulcers. Blood may reduce the sensitivities of all biopsy-based tests.

Feedforward Variable Structural Proportional-Integral-Derivative for Temperature Control of Polymerase Chain Reaction

To track the rapidly changing temperature profiles of thermal cycling of polymerase chain reaction （PCR） accurately, an innovative feedforward variable structural proportional-integral-derivative （FVSPID） controller was developed. Based on the step response test data of the heat block, a reduced first order model was estabfished at different operating points. Based on the reduced model, the FVSPID controller combined a feedforward path with the variable structural proportional-integral-derivative （PID） control. The modified feedforward action provided directly the optimal predictive power for the desired setpoint to speed up the dynamic response. To cooperate with the feedforward action, a variable structural PID was applied, where the P mode was used in the case of the largest errors to speed up response, whereas the PD mode was used in the case of larger errors to suppress overshoot, and finally the PID mode was applied for small error conditions to eliminate the steady state offset. Experimental results illustrated that compared to the conventional PID controller, the FVSPID controller can not only reduce the time taken to complete a standard PCR protocol, but also improve the accuracy of gene amplification.