Establishment of platelet donor gene bank involving specific antigen HPA 1-6/10/15/21 by real-time fluorescent PCR / 中国输血杂志

【Objective】 To research the genetic polymorphism of HPA 1-6/10/15/21 in platelet donors in Zhongshan area, and establish a gene bank of platelet donors with HPA locus. 【Methods】 The HPA 1-6/10/15/21 system genotyping was performed by Real time fluorescent PCR combined with TaqMan probe technology on 192 platelet donors in Zhongshan area, and the genotype frequency and gene frequency were calculated. 【Results】 Only HPA-aa genotype was found within HPA-4/10, and no allele HPA-b had been detected. The majority of HPA-1, 2, 5, 6 and 21 genotypes were aa. HPA-3 and HPA-15 showed high heterozygosity, with genotype frequency of 0.307 3, 0.494 8 and 0.197 9 for HPA- 3aa, HPA-3ab and HPA-3bb, while 0.270 8, 0.505 2 and, 0.224 0 for HPA -15aa, HPA-15ab and HPA-15bb, respectively. 【Conclusion】 The distribution characteristics of HPA 1-6 /10/15/21 of platelet donors in Zhongshan shows regional differences compared with similar researches from other regions. The establishment of HPA gene bank is helpful to avoid alloimmunization caused by incompatible platelet transfusion.

High-risk human papillomavirus in Turkish patients with clinically suspicious cervical lesions analyzed by multiplex-PCR

Background & objectives: Human papillomavirus (HPV) infection is known to be the main cause of cervical cancer. This study aimed to determine the prevalence of high-risk HPV genotypes in smear specimens taken from women who had normal or abnormal cytology using a multiplex PCR method. Methods: The study included 270 women aged between 19 and 69 yr with or without suspicious cervical abnormalities. A Pap smear sample from each patient was cytologically examined, and HPV typing was performed using a multiplex fluorescent PCR method. Those who were high-risk HPV positive and had a normal or abnormal cytology were further evaluated by colposcopy and biopsy. Results: The total HPV positivity was 43 per cent (116/270). HPV positivity in the patients with an abnormal cytology was 77 per cent (33/43), whereas it was only 37 per cent (83/227) in women with normal cytology, which showed a significant difference (P<0.05). HPV positivity was also related to the age group when all the subjects were considered (P<0.05), and the highest prevalence of HPV infection was in the 30-39 yr age group. High-risk HPV types 16, 18, 31, 35, 51 and 56 were more common in the normal cytology patients, whereas high-risk HPV types 16, 31, 35, 45, 58 and 68 were commonly found in the abnormal cytology patients. Interpretation & conclusions: The determination of high-risk HPV genotypes in women with clinically suspicious cervical lesions should be conducted during an annual follow-up, irrespective of a normal or abnormal cytology by the age of 30 years or above.

Phylogenetic relationship analysis of common medicinal submetals and multiple real-time fluorescence PCR identification of Fritillariae Cirrhosae Bulbus / 中草药

Objective To investigate the feasibility of multiple real-time PCR for the detection of Fritillariae Cirrhosae Bulbus and adulterants. Methods Based on the analysis of interspecies variation, genetic distance and phylogenetic relationship of ITS, psbA-trnH, rbcL and matK gene sequences, the genes with fast evolution rate, big interspecies variation and small intraspecies variation were selected as target genes. Fritillariae Cirrhosae Bulbus and adulterants specific primers and Taqman probes were designed to establish a multiplex real-time PCR assay. Methods were evaluated by comparison of specificity, sensitivity and mixed sample detection and sequencing. Results The ITS and psbA-trnH mutations were higher than rbcL and matK, and rbcL and matK were significantly lower than ITS and psbA-trnH genes by genetic distance analysis. And the sensitivity of the establish multiple real-time PCR using ITS as the target gene was 0.01 ng. Four samples of adulterants were detected in 18 samples, and the results were consistent with the results of NJ tree clustering analysis. Conclusion Based on the IIS region sequence as the target gene to establish multiple real-time fluorescence PCR detection method can successfully identify Fritillariae Cirrhosae Bulbus and its counterfeit goods, which provides a new basis for the authenticity of identification.

Identification of Ophiocordyceps sinensis and its adulterants based on portable and CFX96 real-time fluorescent PCR systems / 药学学报

The molecular identification of Ophiocordyceps sinensis and its adulterants was carried out by real-time fluorescent PCR with TaqMan probe. Genomic DNA was extracted from 100 samples of Ophiocordyceps sinensis and its adulterants. MEGA 7.0 software was used for comparative analysis to define the variable sites between Ophiocordyceps sinensis and its adulterants according to the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). A set of specific primers and TaqMan probe were designed using Primer Premier 6.0 software, and sensitivity and specificity studies were performed on two different real-time fluorescent PCR systems (Genesig q16 and Bio-Rad CFX96). The sensitivity study showed that the detectable DNA template concentration of Ophiocordyceps sinensis for the real-time fluorescent PCR was 0.016 ng·μL-1 in the Bio-Rad CFX96 system and 15.527 ng·μL-1 in the Genesig q16 system, respectively. Meanwhile, this method had good specificity for Ophiocordyceps sinensis on Genesig q16 and Bio-Rad CFX96 systems, so Ophiocordyceps sinensis could be clearly distinguished from Ophiocordyceps nutans, Cordyceps gunnii, Cordyceps militaris, Cordyceps cicadae, Cordyceps liangshanensis, Cordyceps gracilis. Our results indicate that real-time fluorescent PCR with TaqMan probe can be used to accurately identify Ophiocordyceps sinensis from its adulterants. This provides a technical method that has wide applications for market management and quality control of Chinese materia medica.

Prenatal diagnosis of an unbalanced translocation between chromosome Y and chromosome 15 in a female fetus

We report the prenatal diagnosis of an unbalanced translocation between chromosome Y and chromosome 15 in a female fetus. Cytogenetic analysis of parental chromosomes revealed that the mother had a normal 46,XX karyotype, whereas the father exhibited a 46,XY,der(15)t(Y;15) karyotype. We performed cytogenetic analysis of the father's family as a result of the father and confirmed the same karyotype in his mother and brother. Fluorescence in situ hybridization and quantitative fluorescent-polymerase chain reaction analysis identified the breakpoint and demonstrated the absence of the SRY gene in female members. Thus, the proband inherited this translocation from the father and grandmother. This makes the prediction of the fetal phenotype possible through assessing the grandmother. Therefore, we suggest that conventional cytogenetic and molecular cytogenetic methods, in combination with family history, provide informative results for prenatal diagnosis and prenatal genetic counseling.

Differences of Nuc-mecA Gene Carriage in the Noses among Different Altitudes in High Humidity District / 现代检验医学杂志

Objective To investigate the carriage of nuc-mecA gene among different altitudes in high humidity district,provid-ed guiding data for prevention of staphylococcus aureus and drug-resistant bacteria,standardizing the usage for antibiotics. Methods The nose swabs were collected in different altitudes:1 000 m,1 200 m and 1 400 m,nuc-mecAgene was confirmed by multi-channel real-time PCR.Results The carrier of nuc gene in the noses were 4.878%,2.899% and 7.143%,in 1 000 m,1 200 m and 1 400 m respectively,and there were no statistical significant among the altitudes (P>0.05).The carrier of mecA gene were 14.634%,31.884% and 41.837% in the 1 000 m,1 200 m and 1 400 m respectively,the difference showed statistical significe (P0.05).Conclusion The carrier of mecA gene in noses was increased with the increasing of the altitude. The residents who living at higher altitude should keep the colonization sites of pathogens clean,and needed timely medical when got sick,shouldn’t abuse the antibiotics without authorization.Medical staff should rational use of antibiotic drugs,a-voided overusing of antibiotics and overtreatment.

Establishment and preliminary application of an assay for the detection of Torque teno sus virus ;strains / 中华微生物学和免疫学杂志

Objective To establish an assay for the detection of Torque teno sus virus ( TTSuV) strains and to analyze its preliminary application to biologics.Methods Primers and probe were designed according to the conserved sequences.A fluorescent PCR assay for the detection of TTSuV strains was estab-lished.Several parameters including the specificity, linearity, accuracy, sensitivity and anti-interference of the established assay were verified.The fluorescent PCR assay was performed to detect the samples of por-cine blood, cell substrate and rotavirus vaccine.The porcine blood samples positive for TTSuV strains were further genotyped.Results The established fluorescent PCR assay was confirmed to have high specificity as no cross-reactions with parvovirus virus of various species, SV40 and porcine circovirus strains were detec-ted.The linear range of the assay was 1×109-1×103 copies/μl for TTSuV1 genotype and 1×109-1×102 cop-ies/μl for TTSuV2 genotype with a R2 value more than 0.993.The sensitivity of the fluorescent PCR assay was 1×103 copies/μl for TTSuV1 genotype and 1×102 copies/μl for TTSuV2 genotype.The intra-and inter-CVs were both less than 7%in Ct values and less than 25% and 45% respectively in copies.No interfer-ence was found in the detection of TTSuV nucleic acids from cell samples.8 out of 20 porcine blood samples were positive for TTSuV strains, among which one sample was positive for TTSuV1 genotype, four samples were positive for TTSuV2 genotype and the rest were positive for both TTSuV1 and TTSuV2 genotypes.Com-pared with the reference strain, strains genotyped as TTSuV1 and TTSuV2 were respectively shared 98%-99%and 98%homologies in sequences.All of the cell substrate and rotavirus vaccine samples detected by the fluorescent PCR assay were negative for TTSuV strains.Conclusion The fluorescent PCR assay for the detection of TTSuV was established successfully, the application of which would further improve the safety of biologics.

Analysis of 75 cases of suspected type A influenza results by real-time fluorescent quantitative PCR test / 国际检验医学杂志

Objective To analyze the detection results of 75 suspected type A influenza cases and to understand the distribution situation of influenza patients to provide the guidance for its early prevention and control .Methods The nasopharyngeal swabs specimens from 75 patients with suspected type A influenza were detected for understanding the viral infection situation by real‐time fluorescent quantitative PCR .Results In the specimens of 75 suspected patients ,13 cases with type A influenza virus were detected out ,in which 9 cases were the H1N1 subtype ,mean age > 50 years old .The temporal distribution was 3 cases in March 2013 ,1 case in April ,7 cases in January 2014 and 2 cases in February .No H7N9 subtype was detected out .Conclusion Real‐time fluores‐cent quantitative PCR has the characteristics of rapidness and high accuracy .The winter is the high onset season of flu ,elderly pa‐tients are susceptible to influenza virus .The monitoring and early treatment should be strengthened .

PCR Multiplex fluorescente para detecção de bactérias em sêmen bovino / Fluorescent Multiplex PCR for detection of bacteria in semen bovine

Este estudo teve como objetivo avaliar o limiar de detecção da técnica de PCR multiplex fluorescente aliada a eletroforese capilar na detecção de agentes infecciosos em amostras de sêmen experimentalmente contaminadas com concentrações decrescentes das bactérias Brucella abortus, Leptospira interrogans sorovar pomona, Campylobacter fetus e Haemophilus somnus. Amostras de sêmen bovino foram experimentalmente contaminadas com concentrações decrescentes de bactérias obtidas através de diluições seriadas na base 10 de modo a obter-se amostras contendo desde 1 vez até 10-7 bactérias/mL a partir da concentração inicial de Leptospira pomona, Brucella abortus, Campylobacter fetus e Haemophilus somnus. As diluições foram efetuadas individualmente para cada bactéria, bem como nas diferentes concentrações necessárias para a padronização do teste de multiplex PCR. As extrações de DNA de todas as soluções contendo espermatozóides e bactérias analisadas no presente estudo foram realizadas segundo protocolo descrito por Heinemann et al. (2000). Os produtos de PCR multiplex foram avaliados por eletroforese em gel de poliacrilamida 8% e separação eletroforética por sistema capilar em equipamento automático de análise de fragmentos de DNA MegaBace. Observou-se a amplificação de fragmentos de 193pb, 330pb, 400pb e 415pb a partir do DNA de B. abortus, L. pomona, H. somnus, C. fetus, respectivamente. Na análise por eletroforese capilar de produtos da PCR multiplex do DNA para detecção simultânea dos quatro patógenos observou-se a sinal de positividade até a diluição de 10-3 bactérias/mL vezes da concentração inicial da solução estoque de cada bactéria. A técnica de PCR multiplex aliada à eletroforese capilar foi usada pela primeira vez para o diagnóstico direto de quatro bactérias patogênicas no sêmen, demonstrando ser um método rápido na detecção de bactérias causadoras de doenças reprodutivas.

This study aimed to evaluate the threshold of detection of fluorescent multiplex PCR coupled with capillary electrophoresis for detection of infectious agents in semen samples from experimentally infected with decreasing concentrations of the bacteria Brucella abortus, Leptospira interrogans serovar pomona, Campylobacter fetus and Haemophilus somnus. Samples of bovine semen were experimentally infected with decreasing concentrations of bacteria obtained from serial dilutions in the base 10 so as to obtain samples containing a long time until 10-7 bacteria/mL from the initial concentration of Lepstospira pomona, Brucella abortus, Haemophilus somnus and Campylobacter fetus. The dilutions were made individually for each bacterium, as well as in different concentrations needed to standardize the multiplex PCR test. DNA extractions of all solutions containing sperm and bacteria analyzed in this study were performed according to protocol described by Heinemann et al. (2000). The multiplex PCR products were analyzed by electrophoresis on 8% polyacrylamide gel and capillary electrophoretic separation system for automated equipment in the analysis of DNA fragments MegaBACE. We observed amplification of fragments of 193pb, 330pb, 415pb and 400bp from the DNA of B. abortus, L. pomona, H. somnus, C. fetus respectively. The analysis by capillary electrophoresis of multiplex PCR products of DNA for simultaneous detection of the four pathogens was observed by detecting the dilution of 10-3 bacteria / mL times the initial concentration of the stock solution of each bacterium. The multiplex PCR coupled with capillary electrophoresis was first used for the direct diagnosis of four pathogenic bacteria in semen, proving to be a rapid method to detect bacteria that cause reproductive disorders.

Expression of Toll-like receptors in the lesions of condyloma acuminatum / 中华微生物学和免疫学杂志

Objective To investigate the expression of Toll-like receptors(TLRs) in condyloma acuminatum(CA) lesions and their possible roles in the pathogenesis of CA. Methods The expressions of TLR1-10 mRNA level in the lesions of CA and in the cervix scrape cells from the patients with human papillomavirus(HPV) negative chronic cervicitis were detected by real-time quantitative fluorescent PCR. HPV typing was detected by HPV GenoArray test kit. Results Low-risk HPV type 6 and type 11 were the most prevalent types in the forty CA cases with positive rate of 77.5% and 55% respectively. 55% CA patients were found infected with more than two types of HPV. 35% CA patients were concurrently infected with high-risk HPV. The expressions of TLR3, 7, 8 mRNA were higher than other TLRs and the expression of TLR9 mRNA was lower than others in the lesions of CA. No significant differences of the TLR1-10 mRNA levels were found between HPV6 and HPV11 positive CA lesions, so did it between low-risk and high-risk HPV concurrent infected CA lesions. The expressions of TLR1-3, TLR5-8, TLR10 mRNA, especially TLR2, TLR7 and TLR8 in the lesions of CA were significantly higher than that in cervix scrape cells of HPV negative chronic cervicitis. There were no significant differences of TLR4 and TLR9 mRNA levels between the two groups. Conclusion There were higher expressions of some TLRs (3, 7, 8) and lower expression of TLR9 in the lesions of CA. Compared with HPV negative chronic cervicitis, the expressions of TLR1-3, TLR5-8, TLR10 mRNA in the lesions of CA were up-regulated. The expression profile of TLRs in different type of HPV infected CA lesions had no significant differences. Our results suggested that the expression profile of TLRs in CA may be associated with the HPV infection. Whether it was associated with the immune escape mechanism and persistent infection of HPV need further demonstration.

Carrier detection and prenatal diagnosis of hemophilia A in a Korean population by fluorescent pcr analysis of dinucleotide repeats in intron 13 and intron 22 of the factor VIII gene / 대한산부인과학회지

OBJECTIVE: To set up the methodology for fluorescent PCR analysis of intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene, and to identify the usefulness of intron 13 and intron 22 microsatellite polymorphism for the carrier detection and prenatal diagnosis of hemophilia A in the Korean population. METHODS: Intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene were analyzed in 30 unrelated Korean mothers of patients with severe hemophilia A using fluorescent PCR. RESULTS: Analysis of intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene was feasible by the fluorescent-PCR method. The expected heterozygosity rates of intron 13 and intron 22 polymorphisms of the factor VIII gene were 67% and 34%, respectively. Combined analysis of intron 13 and intron 22 polymorphisms revealed heterozygous patterns in 16 (53%) of 30 mothers studied. Using linkage analysis with intron 13 and intron 22 polymorphisms, we have attempted three cases of carrier detection and one cases of prenatal diagnosis in two families of patients with severe hemophilia A. CONCLUSION: These results suggest that flourescent-PCR analysis of the intron 13 and intron 22 microsatellite polymorphisms within the factor VIII gene is very useful in the carrier detection and prenatal diagnosis of hemophilia A in the Korean population.

Carrier detection and prenatal diagnosis of hemophilia A in a Korean population by fluorescent pcr analysis of dinucleotide repeats in intron 13 and intron 22 of the factor VIII gene / 대한산부인과학회지

OBJECTIVE: To set up the methodology for fluorescent PCR analysis of intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene, and to identify the usefulness of intron 13 and intron 22 microsatellite polymorphism for the carrier detection and prenatal diagnosis of hemophilia A in the Korean population. METHODS: Intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene were analyzed in 30 unrelated Korean mothers of patients with severe hemophilia A using fluorescent PCR. RESULTS: Analysis of intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene was feasible by the fluorescent-PCR method. The expected heterozygosity rates of intron 13 and intron 22 polymorphisms of the factor VIII gene were 67% and 34%, respectively. Combined analysis of intron 13 and intron 22 polymorphisms revealed heterozygous patterns in 16 (53%) of 30 mothers studied. Using linkage analysis with intron 13 and intron 22 polymorphisms, we have attempted three cases of carrier detection and one cases of prenatal diagnosis in two families of patients with severe hemophilia A. CONCLUSION: These results suggest that flourescent-PCR analysis of the intron 13 and intron 22 microsatellite polymorphisms within the factor VIII gene is very useful in the carrier detection and prenatal diagnosis of hemophilia A in the Korean population.

Detection of human papilloma virus 16/18 by the quantitative fluorescent PCR / 中华微生物学和免疫学杂志

Objective To study the relation between the HPV6/18 virus infection and the development of pathological changes of cervix. Methods The number of HPV16/18 DNA copies and the expression rate of HPV16/18 E7 mRNA in the pathological cervix were examined by the quantitative fluorescent PCR combined with pathological diagnosis and immunohistochemistry staining. Results The HPV16 infection rates in chronic cervicitis group were much lower (7.4%) than that in the cervical intraepithelial neoplasia (CIN) groups and the cervical cancer group (69.6% and 72.7%), respectively. Statistical analysis showed that the difference of HPV16 DNA copies was not significant between the chronic cervicitis group and CIN groups. In contrast to the above mentioned result, the number of HPV DNA copies between the CIN groups and the cervical cancer group was significantly different. The HPV16 E7 gene expression rates in CIN Ⅰ, Ⅱ, Ⅲ and cervical cancer groups were 0,37.5%,42.9%,63.6%, respectively. Conclusion Ins more common than that with HPV18. The number of HPV16 DNA copies in cervical cancer tissues is markedly higher than that in CIN Ⅱ, Ⅲ groups. The HPV16 E7 mRNA expression is significantly increased in the cervical cancer, and it is more closely correlated to this pathological changes. The quantitative fluorescent PCR can be used to reflect the activity of HPV, and it is a useful method for the screening examination of HPV and for the early diagnosis and treatment of cervical caner.

Placental chimerism in early human pregnancy.

Background0 : Human chimerism is rare and usually uncovered through investigations of ambiguous genitalia or blood grouping or prenatal diagnosis. Most of the publications on placental chimerism are mainly case reports. There is no systematic search with sensitive techniques for placental chimerism in human. Aim0 : This study was aimed to asses placental chimerism through two sensitive molecular techniques i.e., interphase fluorescent in situ hybridization and quantitative fluorescent PCR. Material and methods0 : Placental chimerism was analyzed using X & Y dual color fluorescent in-situ hybridization onto 154 placentae from natural conceptions, obtained at termination of pregnancy between 7 to 16 weeks of gestation. Results0 : Three cases of placental sex chromosome chimerism were observed (1.95%). Exclusion of maternal contamination and diagnosis was confirmed later by quantitative fluorescent PCR. Conclusion0 : This finding indicates that placental chimerism in early human pregnancy is not rare.

Efficacy of Duplex-nested PCR and Fluorescent PCR in the Preimplantation Genetic Diagnosis for Duchenne Muscular Dystrophy / 대한불임학회지

OBJECTIVE: Preimplantation genetic diagnosis (PGD) is reserved for couples with a risk of transmitting a serious and incurable disease, and hence avoids the undesirable therapeutic abortion. In this study, we evaluated the efficacy of PGD for Duchenne muscular dystrophy (DMD) cases by the fluorescent PCR with polymorphic linked markers and the conventional duplex-nested PCR methods. METHODS: Biopsy of one or two blastomeres was done from the embryos fertilized by ICSI on the third day after fertilization. We performed two cases of PGD-DMD by the duplex-nested PCR for the causative mutation loci and the SRY gene on Y chromosome. The triplex fluorescent PCR for the mutation loci, the SRY gene and the polymorphic microsatellite marker on X chromosome was applied for two cases of PGD-DMD. RESULTS: By the duplex-nested PCR, successful diagnosis rate was 95.5% (21/22), but we could not discriminate the female embryos whether normal or carrier in this X-linked recessive disease. However, the triplex fluorescent PCR method showed 100% (27/27) of successful diagnosis rate, and all female embryos (n=17) were distinguished normal (n=10) from carrier (n=7) embryos. Unaffected and normal embryos were transferred into mother's uterus after diagnosis. A healthy normal male was achieved after PGD with the duplex-nested PCR method and a twin, a male and a female, were delivered with triplex fluorescent PCR method. The normality of dystrophin gene was confirmed by amniocentesis and postnatal genetic analysis in all offsprings. CONCLUSION: The fluorescent PCR with polymorphic marker might be useful in improving the specificity and reliability of PGD for single gene disorders.

Microsatellite Instability in Korean Hepatocellular Carcinoma using Fluorescent - PCR / 대한암학회지

PURPOSE: Hepatocellular carcinoma (HCC) is one of the most common cancers in many parts of the world, however the molecular mechanisms underlying liver cell transformation remain obscure. The instability of microsatellite sequences dispersed in the genome has been linked to a deficiency in cellular mismatch repair. This phenotype has been frequently observed in various human neoplasms and is regarded as a major factor in tumorigenesis. To investigate cumulative genetic changes related with apoptosis during development and progression of HCC, we examined DNAs isolated from 12 Korean HCCs and their adjacent non-tumorous parts to look for evidence of microsatellite instability (MSI). MATERIALS AND METHODS: Twelve microsatellite loci (D6S271, D6S426, D13S153, D13S263, D17S849, D17S938, D17S945, D18S474, D18S64, D19S420, D.19S418 and D19S210) were amplified by PCR from 12 Korean HCCs, and analyzed using an automated DNA analyzer. RESULTS: The high percentages of the MSI were found for the loci of D6S426 (33.3%) and D17S945 (25.0%). The related genes with high frequency of MSI were noted in the wafl (41.7%) and p53 (25.0%). From this study, fifty eight percent of HCCs (7/12) showed MSI with at least one marker. CONCLUSION: This results suggest that the analysis of MSI in HCC might be useful for identifying genes whose loss of function contributes to the development of liver cancer. Furthennore, this method may give a more rapid and accurate sizing of the PCR products of microsatellite; making the routine assessment of MSI possible in many clinical fields.

Detection and Identification of Tilletia controversa K?hn by Real-time fluorescent PCR / 微生物学通报

By analysing the ribosome gene(rDNA) sequences in externally transcribed spacer(ETS) of wheat T.controversa(TCK) and its similar spacies T.caries(TCT)and T.foetida(TFL).The special sequences of TCK's ETS have been found,.And designed Taqman probe according to the special sequences,TCK has been successfully detected by using Real-time Fluorescent PCR.