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.

Primary application of a real-time quantitative polymerase chain reaction for the detection of human breast cancer related novel gene-Metadherin expression

Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to explore the relationship between expression of Metadherin gene in the patients peripheral blood and the clinic-pathological features in breast cancer. Methods:Real-time fluorescence quantitative polymerase chain reaction was employed to determine the expression level of Metadherin gene in 80 peripheral blood samples of breast cancer patients and healthy donors. Results:The expression of Metadherin gene in breast cancer patients peripheral blood were positive,in which 34 breast cancer patients were highly expressed,accounting for 55.7%,while the expression of Metadherin gene in normal females peripheral blood were negative,there was statistical significance (Ratio = 2.02±0.81,P < 0.05); Ratio of the Metadherin expression in breast cancer patients peripheral blood and the glyceraldehyde-3-phosphate dehydrogenase expression was 1.15 ± 0.36. REST software analysis showed that the expression of Metadherin gene was significantly up-regulated in breast cancer. Conclusion:The SYBR Green I quantitative real-time polymerase chain reaction method can successfully detect the expression level of Metadherin gene. Expression level of Metadherin gene in breast cancer patients peripheral blood is closely related to survival,and it maybe involved in the development of breast cancer and used as an indicator of prognosis.

Real-time Fluorescence PCR Method for Detection of Burkholderia glumae from Rice

Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1：100.

Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus

The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.

Rapid prenatal diagnosis of trisomy 21 by fluorescent quantitative multiplex polymerase chain reaction

Trisomy 21, also named Down syndrome was the most frequent autosomal aneuploidy and the most common cause of mental retardation. Fifty percent patients had congenital heart malformation. Every 20 minutes one case of trisomy 21 was born, and the incidence rate was 1 in 600 to 800 newborns in China.1 In two thirds of cases with trisomy 21, there was a spontaneous abortion, so the actual incidence was higher than that obtained postnatally.

Most common SLC25A13 mutation in 400 Chinese infants with intrahepatic cholestasis

AIM:To establish the real time fluorescence polymerase chain reaction(RT-PCR) with dual labeled probes for fast detection of SLC25A13 gene mutation 851del4.METHODS:Four hundred infants(< 1 year of age) with unexplained intrahepatic cholestasis from 18 provinces or municipalities in China were enrolled in this study for detecting their SLC25A13 gene mutation 851del4.Suitable primers and fluorescence-labeled probes for detecting SLC25A13 gene mutation 841del4 were designed.Normal and mutant sequences were detected by PCR with two fluorescence-labeled probes.After a single RT-PCR,results were obtained by analyzing the take-off curves.Twenty-four positive and 14 negative samples were retested by direct sequencing.RESULTS:Eight homozygous and 30 heterozygous mutations were detected in 46 mutant alleles with a 851del4 mutation rate of 5.8%(46/800).Twenty-six and 20 mutant alleles were observed respectively,in 474 and 242 alleles from the intermediate and southern areas of China.No mutant allele was detected in 84 alleles from northern China.Twenty-four positive samples including 4 homozygous and 20 heterozygous mutations,and 14 negative samples were retested by direct sequencing,which confirmed that the accuracy of RTPCR was 100%.CONCLUSION:RT-PCR can detect the mutation 851del4 in infants with intrahepatic cholestasis with an accuracy of 100%.

Association of Fusobacterium nucleatum infection with colorectal cancer in Chinese patients

AIM: To investigate Fusobacterium nucleatum (F. nucleatum) abundance in colorectal cancer (CRC) tissues and its association with CRC invasiveness in Chinese patients.METHODS: The resected cancer and adjacent normal tissues (10 cm beyond cancer margins) from 101 consecutive patients with CRC were collected. Fluorescent quantitative polymerase chain reaction (FQ-PCR) was applied to detect F. nucleatum in CRC and normal tissues. The difference of F. nucleatum abundance between cancer and normal tissues and the relationship of F. nucleatum abundance with clinical variables were evaluated. Fluorescence in situ hybridization (FISH) analysis was performed on 22 CRC tissues with the highest F. nucleatum abundance by FQ-PCR testing to confirm FQ-PCR results.RESULTS: The median abundance of F. nucleatum in CRC tissues [0.242 (0.178-0.276)] was significantly higher than that in normal controls [0.050 (0.023-0.067)] (P < 0.001). F. nucleatum was over-represented in 88/101 (87.1%) CRC samples. The abundance of F. nucleatum determined by 2-ΔCT was significantly greater in tumor samples [0.242 (0.178, 0.276)] than in normal controls [0.050 (0.023, 0.067)] (P < 0.001). The frequency of patients with lymph node metastases was higher in the over-abundance group [52/88 (59.1%)] than in the under-abundance group [0/13 (0%)] (P < 0.005). No significant association of F. nucleatum with other clinico-pathological variables was observed (P > 0.05). FISH analysis also found more F. nucleatum in CRC than in normal tissues (median number 6, 25th 3, 75th 10 vs 2, 25th 1, 75th 5) (P < 0.01).CONCLUSION: F. nucleatum was enriched in CRC tissues and associated with CRC development and metastasis.

Establishment of a new quantitative detection approach to adefovir-resistant HBV and its clinical application

AIM:To establish the more feasible and sensitive assessment approach to the detection of adefovir (ADV) resistance-associated hepatitis B virus (HBV) quasispecies.METHODS: Based on the characteristics of rtA181V/T and rtN236T mutations, a new approach based on real-time fluorescent quantitative polymerase chain reaction (RT-PCR) was established for the detection of ADV-resistant HBV quasispecies, total HBV DNA, rtA181 and rtN236 mutations in blood samples from 32 chronic hepatitis B (CHB) patients with unsatisfactory curative effect on ADV and compared with routine HBV DNA sequencing.RESULTS: Both the sensitivity and specificity of this new detection approach to ADV-resistant HBV quasispecies were 100%, which were much higher than those of direct HBV DNA sequencing. The approach was able to detect 0.1% of mutated strains in a total plasmid population. Among the 32 clinical patients, single rtA181 and rtN236T mutation and double rtA181T and rtN236T mutations were detected in 20 and 8, respectively, while ADV-resistant mutations in 6 (including, rtA181V/T mutation alone in 5 patients) and no associated mutations in 26.CONCLUSION: This new approach is more feasible and efficient to detect ADV-resistant mutants of HBV and ADV-resistant mutations before and during ADV treatment with a specificity of 100% and a sensitivity of 100%.

Expression of Twist Gene in Human Hepatocellular Carcinoma Cell Strains of Different Metastatic Potential

In order to investigate the role of Twist gene in the metastasis of hepatocellular carcinoma （HCC）, total RNA was respectively extracted from three HCC cell strains with different metastatic potentials, HepG2, MHCC-97L and MHCC-97H. The first strand cDNA was synthesized by reverse transcription, which was then used as template to perform fluorescent quantitative polymerase chain reaction （FQ-PCR）. The quantity of Twist gene expression was normalized by that of the housekeeping gene, GAPDH for each sample. ANOVA was used to estimate the relationship between Twist gene and metastasis potential of HCC. The results showed that the normalized initial cDNA concentrations of Twist gene in HepG2, MHCC-97L and MHCC-97H were （9.45±0.25）×10^-4, （ 1.82±0.41 ）× 10^-3, （3.06±0.62）×10^-3, respectively. FQ-PCR revealed significan, t differences in the expression level of Twist among HCC cell strains with different metastatic potentials. It was concluded that high expression level of Twist was closely associated with more aggressive behaviors of HCC. Twist provides a novel indicator for HCC metastasis.

Prenatal diagnosis of Down syndrome using cell-free fetal DNA in amniotic fluid by quantitative fluorescent polymersase chain reaction

Backgroud Amniotic fluid （AF） supernatant contains cell-free fetal DNA （cffDNA） fragments.This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis,which could be combined with simple quantitative fluorescent polymerase chain reaction （QF-PCR） to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome.Methods AF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes.Peripheral blood samples of the parents were collected at the same time.Short tandem repeat （STR） fragments on chromosome 21 were amplified by QF-PCR.Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks.Results The sensitivity of the assay for the aneuploid was 93％ （26/28; confidence interval,CI：77％-98％） and the specificity was 100％ （26/26; CI：88％-100％）.The determination rate of the origin of the extra chromosome was 69％.The sensitivity and the specificity of the assay in the euploid were 100％ （27/27）.Conclusions Trisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant.This karyotype analysis method greatly reduces the requirement for the specimen size.It will be a benefit for early amniocentesis and could avoid pregnancy complications.The method may become an ancillary method for prenatal diagnosis of trisomy 21.

Cx40 mRNA expression in crista terminalis and left atrium of patients with rheumatic heart disease associated chronic atrial fibrillation

Objectives： To explore possible mechanisms of connexin40 （Cx40） remodeling by detecting Cx40 mRNA expression of the crista terminalis and left atrium （LA） in patients with rheumatic heart disease （RHD） associated chronic atrial fibrillation （AF）. Methods： Twenty patients were enrolled in this study, who underwent surgical operation for RHD-associated mitral disease, including l0 with sinus rhythms （rhythm group） and l0 with AF （AF group）. Another 6 patients with non-RHD sinus rhythms were divided into the control group. A small amount of myocardial tissue was cut from the crista terminalis and the LA posterior wall during the valvular replacement operation. Cx40 mRNA expression was assayed by real-time fluorescent quantitation polymerase chain reaction （RT-PCR）. Results： There was no significant difference in Cx40 mRNA expression in the crista terminalis and LA posterior wall between the 3 groups, and there was no significant difference in Cx40 mRNA expression between the crista terminalis and LA within each group. Conclusion： Based on the finding in previous studies that there existed evident remodeling of atrial Cx40 protein in patients with chronic RHD, the results of the present study suggest that the mechanism of Cx40 remodeling probably lies in the post transcriptional level.

Effect of electroacupuncture on expression of NR2B in prefrontal cortex in morphine-withdrawal rats

Objective To observe the effect of electroacupuncture （EA） on learning and memory abilities and expression of N-methyI-D-aspartate receptor subunit （NR2B） in prefrontal cortex in morphine-withdrawal rats and to investigate the molecular biological mechanisms. Methods Thirty-six male SD rats were randomly divided into four groups, namely control group （group A）, model group （group B）, model with acupuncture group （group C） and model with electroacupunture group （group D）, with 9 in each group. All rats except those in group A were subcutaneously injected with morphine hydrochloride injectio on the back with daily dosage increased day by day. Naloxone was given 3 h after the last injection to establish the models of morphinewithdrawal rats. After the models were established, the rats were treated with acupuncture and electroacupuncture respectively at bilateral ＂Shenshu＂ （肾俞 BL 23） and ＂Zusanli＂ （足三里 ST 36） for 15 min per time, once daily for 6 days. Space learning and memory abilities of the rats were measured by Morris water maze, and protein and gene expression levels of NR2B in prefrontal cortex were measured by Western Blot and RT-PCR. Results In place navigation test, the escape latency in group B, group C and group D was significantly prolonged compared with that of group A （P〈0.01）, the escape latency in group C and group D was significantly shortened compared with that of group B （P〈0.01） and the escape latency in group D was significantly shortened compared with that of group C （P〈0.05）; during spatial probe test, the number of times crossing the platform of group B, group C and group D decreased compared with that of group A （P〈0.01）, and compared with group B, the number of times crossing the platform of group C increased and the number of group D significantly increased （P〈0.01）. Decreased protein expression level of NR2B was found in group B when compared with that of group A （P〈0.01）, increased protein expression levels of NR2B were found in group C and group D when compared with that of group B （P〈0.01）, however, the expression level in group D was higher than that in group C （P〈0.01）. mRNA expression level of NR2B in prefrontal cortex in morphine-withdrawal rats decreased （P〈0.05）, however, compared with that of group B, the expression level increased in group D （P〈0.05）, and there was no statistical significance in increased expression level in group C （P〉0.05）. Conclusion Acupuncture and eletroacupunture can improve space learning and memory abilities of merphine-withdrawal rats, with better efficacy of eletroacupuncture than that of acupuncture, the mechanisms of which may be associated with the regulation of NR2B expression in prefrontal cortex.

Dynamic observation of polypide in semen and blood of rabbits infected with Toxoplasma tachyzoites

Toxoplasma gondii （T. gondfi） is one of the intracellular parasitized protozoa and may cause severe medical complications in fetus or immunocompromised individuals. T. gondii existed as tachyzoite during acute stage while as bradyzoite during chronic phase in human cells. To improve understanding of the prevention, diagnosis, and treatment of the disease, it is important to explore the distribution and fluctuation and other biological features of T. gondii in host. The trophozoite had been found in the saliva, blood or urine of the host. Some studies suggested the dynamic changes of circulating antibody and toxoplasma circulating antigen （TCA） either in blood or in urine. T. gondii in tissue or blood cannot be counted exactly under the microscope because it was only several micrometers in size and thus most of the studies were performed qualitatively by mouse inoculation or immunology methods. The quantitative fluorescent polymerase chain reaction （QF-PCR） and its application raised the possibility for dynamic observation of the polypide in the host. In this study, blood and semen were collected from the male rabbit model infected with toxoplasma tachyzoites and T. gondii was detected by QF-PCR quantitatively.