Concordance of identifying the presence or absence of KIR genes by Flow-rSSO and PCR-SBT methods: a comparative study / 中国输血杂志

【Objective】 To study the concordance of identifying the presence or absence of KIR genes using flow reverse sequence-specific oligonucleotide probe (Flow-rSSO) hybridization and sequencing based typing-PCR (PCR-SBT) methods. 【Methods】 A total number of 131 cases of DNA samples from Han population were subjected to identify the presence or absence of all 16 KIR genes by Flow-rSSO method, and then sequenced at coding sequence for all 14 functional KIR genes using our in-house KIR PCR-SBT assay. The concordance of identifying the presence or absence of all functional KIR genes by Flow-rSSO and PCR-SBT was analyzed. Samples with inconsistent initial results were re-tested using the Flow-rSSO commercial kits with different Lot number, and further tested using the PCR-SSP commercial kit. 【Results】 The presence or absence of 14 functional KIR genes for 129 of 131 samples were completely in accordance via the PCR-SBT and Flow-rSSO methods. Two samples, one with 3DL1 negative, the other with both 2DS3 and 2DS5 negative initially-identified by Flow-rSSO, were actually all positive tested by PCR-SBT. Further retest by Flow-rSSO commercial kits with different Lot number and PCR-SSP commercial kit indicated that the two samples were all positive, which agreed well with PCR-SBT results. 【Conclusion】 In this paper, the initial test results of the presence or absence of KIR genes identified by Flow-rSSO for 2 samples were wrong, which indicated the importance of carrying out the quality control for reagents in KIR gene testing.

Shoot rot of Zizania latifolia and the first record of its pathogen Pantoea ananatis in China / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The aquatic grass Zizania latifolia grows symbiotically with the fungus Ustilago esculenta producing swollen structures called Jiaobai, widely cultivated in China. A new disease of Z. latifolia was found in Zhejiang Province, China. Initial lesions appeared on the leaf sheaths or sometimes on the leaves near the leaf sheaths. The lesions extended along the axis of the leaf shoots and formed long brown to dark brown streaks from the leaf sheath to the leaf, causing sheath rot and death of entire leaves on young plants. The pathogen was isolated and identified as the bacterium Pantoea ananatis, based on 16S ribosomal RNA (rRNA) gene sequencing, multilocus sequence analysis (atpD (β-subunit of ATP synthase F1), gyrB (DNA gyrase subunit B), infB (translation initiation factor 2), and rpoB (β‍-subunit of RNA polymerase) genes), and pathogenicity tests. Ultrastructural observations using scanning electron microscopy revealed that the bacterial cells colonized the vascular tissues in leaf sheaths, forming biofilms on the inner surface of vessel walls, and extended between vessel elements via the perforated plates. To achieve efficient detection and diagnosis of P. ananatis, species-specific primer pairs were designed and validated by testing closely related and unrelated species and diseased tissues of Z. latifolia. This is the first report of bacterial sheath rot disease of Z. latifolia caused by P. ananatis in China.

HLA- A & B Association in Psoriasis in Tamil Speaking South Indian Population.

Psoriasis is an auto-immune skin disease affecting skin, nails and joints. The association of HLA with psoriasis is already established with HLA- C*06 known to be associated strongly with the disease. We wanted to determine the HLA -A & HLA-B pattern and its association with psoriasis in a Tamil speaking ethnic population.METHODSA total of 100 psoriasis patients attending the Dermatology OPD at SRMC were taken up for the study. This was a case control study and hence 100 voluntary blood donors donating at the SRMC Hospital blood bank were taken up for study as controls. Voluntary blood donors are considered as healthy normal individuals and hence chosen as controls. All the 100 patients and 100 controls were typed for HLA (Human Leucocyte Antigen) - A & B by PCR-SSP (Polymerase Chain Reaction-Sequence Specific Primers) and the results were analysed statistically using OpenEpi software (2 X 2 table). The Odds Ratio (OR), p (probability) value, and 95% confidence interval were the statistical tests which were studied.RESULTSHLA-A*02, 24 and HLA-B*35 were found to be strongly associated with psoriasis among Tamil speaking ethnic population.CONCLUSIONSThere are different HLA – A & B alleles associated with psoriasis in Tamil ethnic population in comparison with other ethnic studies

Association of HLA-C 06 with Psoriasis in a Tertiary Care Hospital.

Prevalence of psoriasis is 1-3% in India. HLA-C*06 has been shown to be strongly associated with psoriasis in different ethnic populations. This study was carried out to determine the association of HLA-C in psoriasis patients in a south Indian ethnic population.METHODSA total of 200 samples were included in the study. In all, 100 psoriasis patients and 100 healthy controls were studied. HLA-C typing was done by PCR-SSP method. Results were analysed statistically using open epi software (2 X 2 table). The Odds ratio (OR), p (probability) value, and 95% confidence interval were the statistical tests applied and analysed.RESULTSA total of 14 different HLA-C alleles were identified in both 100 cases and 100 controls. Among the 14 different HLA-C alleles, the alleles which were found to be strongly associated with psoriasis which were statistically significant were both HLA-C*06 and HLA-C*07. HLA-C*06 was found to be present in 52% of the patients and HLA-C*07 was found to be present in 33% of the patients. HLA-C*06 was found to be strongly associated with the disease in 52% of the patients.CONCLUSIONSThis study confirms HLA-C*06 association with psoriasis which is in concordance with other previous studies.

DNA Barcoding Identification of Paecilomyces hepiali and Jinshuibao Capsules / 中国实验方剂学杂志

Objective::Because traditional methods are difficult to identify the fermentation mycelium, DNA barcoding technology was used to quickly identify the raw material strain Paecilomyces hepiali of Jinshuibao capsules and related products. Method::A total of 168 samples of 8 species of P. hepiali and its confusable species were identified by internal transcribed spacer (ITS) sequences, and based on the ITS sequences, P. hepiali specific primers were designed to quickly identify the related products. Result::The length of ITS sequences in 44 P. hepiali samples from different sources was 499 bp and there was no mutation site. It was shown that P. hepiali could be distinguished from 7 confusable species based on ITS sequences. The specific primer (ITS-BF/ITS-BR) of P. hepiali designed by ITS sequences could be amplified to obtain a short fragment of 102 bp in length, which could be used to rapidly identify P. hepiali from other confusable species, and to distinguish relevant products in the market. Conclusion::The rapid identification of P. hepiali and its related products can be achieved through the ITS sequences and specific primers, which provides a reference for the production and quality control of Jinshuibao capsules.

Development and Comparison of Determination of Staphylococcus aureus in Different Processed Products of Angelicae Sinensis Radix Based on Real-time PCR Technique / 中国实验方剂学杂志

Objective:To quantitatively analyze the changes of Staphylococcus aureus in different processed products of Angelicae Sinensis Radix. Method:The real-time fluorescence quantitative polymerase chain reaction method （Real-time PCR） was established to quantitatively analyze S. aureus in Angelicae Sinensis Radix decoction pieces which bought from different producing areas， different enterprises and different storage time. The fluorescence quantitative reaction system was SYBR Premix Ex Taq Ⅱ of 10 μL， each of forward primer and reverse primer （10 μmol·L-1） of 0.8 μL， template/genome DNA of 1 μL， double distilled water of 7.4 μL. The reaction conditions of the fluorescence quantitative amplification curve were pre-denaturing for 30 s at 94 ℃， denaturing for 10 s at 94 ℃， annealing for 12 s at 60 ℃， extensing for 30 s at 72 ℃， cycling 45 times， single-point detection signal at 72 ℃. The melting curve was made from 72 ℃， and the step temperature of 0.5 ℃ was kept for 15 s to collect fluorescence. According to the results of Real-time PCR， representative samples were selected from Angelicae Sinensis Radix decoction pieces for comparison between plate counting method and Real-time PCR. Result:The content of S. aureus in different processed products was sorted by rank of raw Angelicae Sinensis Radix>soil-fried Angelicae Sinensis Radix>wine-processed Angelicae Sinensis Radix. The content of S. aureus was the lowest in the samples from Weiyuan area of Gansu province by comparing with other producing areas. Compared with the retail enterprises， the content of S. aureus in raw products and wine-processed products from production and sale enterprises was lower. Different storage time had certain effect on the content of S. aureus in raw products and wine-processed products， and the content of S. aureus increased with the increase of storage time. The detection results of plate counting method were 3-4 orders of magnitude lower than that of Real-time PCR. Conclusion:The established Real-time PCR is superior to plate counting method in specificity， sensitivity， reliability and reporting period， which can provide an effective method for rapid and accurate quantitative detection of S. aureus in different processed products of Angelicae Sinensis Radix.

Ambiguity results distribution and its solutions of HLA-A, B and DRB1 sequence-based typing / 重庆医学

Objective To investigate the ambiguity results distribution of HLA-A,B and DRB1 gene sequence-base typing in Guangxi population and to propose the way to resolve.Methods HLA-A,B and DRB1 genes of 1 000 donors in the Guangxi branch bank of China'bone marrow bank were genotyped by PCR-SBT,and then the ambiguity results distribution of the three loci was analyzed.The typing ambiguities resultswere resolved by high-resolution polymerase chain reaction-sequence-specific primers(PCR-SSP) and group specific sequencing primer(GSSP) methods,respectively.Results Among 1 000 samples,at least 1 locus in HLA-A,B and DRB1 genes in 96.7％ samples appeared the ambiguity results,in which the proportions of HLA-A,B and DRB1 loci appearing ambiguity results were 65.7 ％,58.8 ％ and 77.2 ％ respectively.For the samples of detected ambiguity results,single using the GSSP method could resolve the ambiguity typing results of 87.37％ HLA-A,93.54％ HLA-B and 60.49％ HLA-DRB1,using high-resolution PCR-SSP could resolve the ambiguity typing results of 12.63 ％ HLA-A,4.76 ％ HLA-B and 15.29 ％ HLA-DRB1,and the rest 1.70 ％ HLA-B and 24.22 ％ HLA-DRB1 ambiguity results were resolved by both GSSP and high-resolution PCR-SSPs method.Conclusion GSSP and high-resolution PCR-SSPs methods have high abilities to solve HLA ambiguity results both locate inside and outside the sequencing region,respectively.GSSP and high-resolution PCR-SSPs methods are supplement for each other,which can effectively resolve the problem of ambiguity results in high resolution HLA typing.

Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains

Abstract In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines.

Rapid molecular identification of saffron (Crocus sativus L.) based on specific PCR / 中国药学杂志

OBJECTIVE: To establish a rapid molecular method for identifying saffron (Crocus sativus L.) and its adulterants by PCR: amplification using specific primers and fluorescent dyes detection. METHODS: The chloroplast barcode was sequenced and analyzed to find the SNPs between saffron and its adulterants. Specific primers were designed for the SNPs, the PCR reaction systems were built and optimized, and fluorescent dyes method was used to identify PCR products. RESULTS: A 421 bp saffron identification band based on the psbA-trnH barcode sequence was screened when 100 × SYBR Green I was added into the optimized PCR product under the following condition; initial denaturation at 90℃ for 1 min, denaturation at 90℃ for 5 s, annealing at 58℃ for 5 s, 26 cycles; the saffron (Crocus sativus L.) showed strong green fluorescence under 365 nm UV lamp whereas adulterants did not. CONCLUSION: Fast site-specific PCR can rapidly identify saffron and its adulteration.

Highly Specific DNA Barcoding Primers of ITS2 for Gastrodia elata / 世界科学技术-中医药现代化

This study aimed to design a pair of primers for amplifying internal transcribed spacer 2 region which was used to identify Gastrodia elata through optimizing of DNA extraction and PCR amplification process. Two sequences are used for research object by amplification of universal primers. Design three pairs of specific primers by Primer Premier 5.0 and select the highest specificity through the study of 22 samples. The results showed that identification efficiency of the primer named TM2F-2R is as high as 90.9% when Annealing Temperature is equal to 54 degrees Celsius. Therefore, TM2F-2R can be used as primers ITS2 sequences of G. elata, this article provides a set of accu-rate and stable identification methods for G. elata in the molecular identification.

Recombination in human leukocyte antigen region in two Asian Indian families.

BACKGROUND: Recombination (crossing over) may generate novel haplotypes that can be beneficial to a population against recently introduced pathogens. It may lead to the generation of new alleles. SETTINGS AND DESIGN: A prospective study at a tertiary care centre. AIM: To report two rare cases of crossing over in HLA region. MATERIALS AND METHODS: Tissue-typing was done by sequence specific primers (SSP) for DR locus and by both SSP and serology for Class I which was reconfirmed on fresh samples. RESULTS: In one patient crossing over had taken place in the region of A locus resulting in inheritance of A*01 instead of expected A*11. In second family crossing over had taken place in region of DRB1 locus and the sibling inherited DRB1*08 instead of DRB1*10. CONCLUSIONS: Possibility of recombination must be considered when interpreting implausible tissue-typing results of families worked up for BMT.

Molecular authentication of the traditional Chinese medicinal plant Angelica sinensis based on internal transcribed spacer of nrDNA / Autentificación molecular de la planta medicinal tradicional china Angelica sinensis basada en el espaciador interno transcrito del ADNnr

Traditionally, the authentication of the traditional Chinese medicines (TCM), Angelica sinensis, is based on slightly different morphological characters and complex compounds. Usually, those methods are simultaneously affected by several factors, leading to subtle and ambiguous results. In this study, the internal transcribed spacer (ITS) regions of A. sinensis and seven other Angelica species used as adulterants were sequenced. A pair of specific primers was designed from the polymorphic ITS regions to distinguish A. sinensis from the adulterants and regional substitutes. These ITS-derived primers amplified approximately 520 bp specific fragments from the adulterants, whereas no products was amplified with the DNA of A. sinensis. We tested eight commercially crude materials purchased in the market by using these specific primers. The result showed that there were four samples adulterating A. sinensis with regional substitutes. This indicated that A. sinensis could be accurately distinguished from the adulterants and regional substitutes. Therefore, the method of molecular authentication based on the ITS sequences may be contributed to raw material production and quality control of A. sinensis.

Association of hemorrhagic fever with renal syndrome and HLA-DRB allele polymorphisms in Han nationality, Hubei province / 中华流行病学杂志

Objective To understand the association of human leukocyte antigen (HLA)-DRB polymorphism and patients diagnosed as hemorrhagic fever with renal syndrome (HFRS). Methods HLA-DR allele polymorphism was detected by PCR-sequence specific primers (PCR-SSP). Hantavirus (HV) typed as Hantaan virus (HTNV) and Seoul virus (SEOV) in patients were detected by RT-heminested PCR. Results The gene frequency of DRB1*0401-0411, *1001 and *1101-1105 in HFRS case group were 3.1%, 2.2% and 15.7% respectively. Compared with control group, it was significant higher in HFRS case group (RR=13.87, 9.72 and 2.00 respectively with Chi-square value as 10.006,6.324 and 6.472 respectively, P<0.05). When compared with HFRS case group, the gene frequency of DRB1*1501-1502, DRB4 and DRB5 in control group were 11.0%, 19.0% and 16.9% respectively, markedly lower than in patients (RR=0.45, 0.58 and 0.23 respectively. Chi-square values were 6.138, 4.583 and 21.076 respectively, P<0.05). There was no significant difference in other HLA-DR gene frequencies. Mixed infection was found in Hubei, with HTNV slightly more than SEOV. Distinct hantaviruses could coexist in either different or the same geographic or ecological zores in Hubei province. Patients with HLA-DRB1*1101-1105 alleles were 81.8%(27/33) infected by HTNV and only 18.2% infected by SEOV, which had significant difference (P<0.05). Conclusion DRB1*0401-0411,*1001 and *1101-1105 were possibly associated with increased susceptibility to HV infection. On the other hand there was an inverse correlation among HFRS, DRB1*1501-1502, DRB4 and DRB5.

Evaluation of two type-specific primers PCR genotyping methods of hepatitis B virus / 中华微生物学和免疫学杂志

Objective To compare and evaluate two type-specific primers PCR genotyping methods of hepatitis B virus ( HBV) which were established by Naito et al ( Naito method) and our lab (new method). Methods The two genotyping methods were applied for detecting the plasmids containing the HBV genomes of genotype A or D or subgenotype B1 or C2 and the plasmids mixed with different proportion of subgenotypes B1 and C2. In addition, the genotypes of 113 serum samples of patients with chronic HBV infection from Shenzhen, Handan and Urumqi cities of China were identified by the two methods, respectively. The results were verified by PCR product based sequencing. Results The sensitivity of the two methods showed no difference when they were applied to detect the plasmids containing the HBV genomes of genotype A or D or subgenotype B1 or C2. While detecting the plasmids mixed with different proportion of subgenotypes B1 and C2, the sensitivity of the new method was superior than that of Naito method. Meanwhile, the specificity of the new method was obviously superior than that of Naito method. Both of the two methods were highly sensitive in identification of HBV genotypes of serum samples with a single genotype. However, the new method showed more sensitive in identification of the B/C mix strains than that of Naito method. The total coincidence rate between the two methods was 83. 2% (94/113), with the discrepancy of 16. 8% (19/113). Fifteen of the 19 inconsistent genotypes by the two methods were selected and their PCR products were sequenced directly. The sequencing results were identical with that of the new methods, but not with that of the Naito method. Conclusion The new method is more sensitive, and its specificity is superior to the Naito method. It could be used for clinical and epidemiological studies on HBV genotype and subgenotype in China.

Multiplex-PCR Approach to Identify Bacillus megaterium and Bacillus cereus Group Applied in Microbial Fertilizers / 微生物学通报

Bacillus megaterium strains are commonly used in microbial fertilizer(MF) . MF products are often contaminated by other B. cereus group members,which have similar phenotype such as Bacillus cereus,B. thuringiensis,B. mycoide. For quality control and safety of MF,a rapid and accurate method is needed to distinguish the strains of Bacillus megaterium from B. cereus group. Based on specific nucleotide sequences of the spoOA genes,2 pairs of species-specific primers were designed and a multiplex-PCR(mPCR) was developed for this purpose. When the optimized mPCR was used to detect the DNAs of 24 reference strains from three genera of Bacillus,Paenibacillus,and Brevibacillus,all B. megaterium strains showed singlefragment of 443 bp and Bacillus cereus group showed a fragment of 411 bp. However,no any amplified product was from the other bacteria. The sensitivity of mPCR was 105 CFU/mL. The mPCR results of 10 isolates of B. megaterium/B. cereus group and 8 products of MF coincided with the biochemical assay. Taken together,our newly developed mPCR assay was species-specific and effective in application. It can be used to detect and identify the strains of B. megaterium and B. cereus group from microbial fertilizer products.

Study of the genetic and polymorphism of human platelet antigens 1—6,15 in unrelated volunteer blood donors,Guangzhou / 中国输血杂志

Objective To study the polymorphism of human platelet antigens among unrelated Guangzhou blood donors with PCR-SSP,in order to provide basic data for population studies and clinical transfusion practice.Methods Blood samples from 706 unrelated blood donors in Guangzhou were genotyped for each of the HPA1—6,15 systems by PCR-SSP.Gene frequencies and genotype frequencies were analyzed by statistical methods.Results HPA-3 and-15 had the greatest heterozygosity with a gene frequency of 0.2918,0.4830,0.2252 for HPA-3a/a,HPA-3a/b,HPA-3b/b,and 0.2691,0.5170,0.2139 for HPA-15a/a,HPA-15a/b,HPA-15b/b.The a/a homozygosity was predominant in HPA-1,-2,-4,-5,with a frequency ranged from 0.9583 to 0.9993,while HPA b/b was not found among them.The frequency of HPA-lb and HPA-4b was very low,which was 0.0028 and 0.0007,respectively.In our study,HPA-1 frequency was significantly different from that of the north Chinese,English,and American Indian(P

Studies of interaction between KIR alleles and the progression of HIV-1 infection in Chinese population / 中华微生物学和免疫学杂志

Objective To investigate the killer cell lg-like receptors (KIR) gene frequency of HIV-1 infected slow progressors(SP) and typical progressors(TP), and to analyze the interaction between KIR alleles and the progression of HIV-1 infection in Chinese population. Methods Eighty-one HIV-1 posi-tive individuals including 43 SPs and 38 TPs were recruited. Carriage of KIR genes was assessed using poly-merase chain reaction sequence-specific primers (PCR-SSP) assays. Results KIR2DS3 gene frequency was significantly lower in SP group (3.6%) than that in TP group (14.2%), P =0. 018 ,OR =0. 210,95% CI =0.053-0.833. The number of activating KIR genes was less in SP group than that in TP group, but was not significant (P = 0. 208). Conclusion Lower KIR2DS3 gene frequency may potentially be associated with slower progression to AIDS in Chinese population.

Effects of Human Platelet Antigen Genotyping on Platelet Engraftment and Graft-Versus-Host Disease after Hematopoietic Stem Cell Transplantation / 대한진단검사의학회지

BACKGROUND: Rapid platelet engraftment has several economic benefits by reducing the cost of supportive therapy as well as reducing the risk of fatal bleeding due to severe thrombocytopenia. Based on these considerations, we genotyped human platelet alloantigens (HPA) to evaluate the effect of minor transplantation antigen mismatches on the rate and speed of platelet recovery and clinical outcome of transplantation. METHODS: Thirty-five patients with various hematologic diseases transplanted between January 2001 and August 2004 were included. Genomic DNA was isolated from peripheral blood of donor-recipient pairs before transplantation. HPA-1, -2, -3, -4, -5, and -6 genotyping was performed by poly-merase chain reaction (PCR)-sequence specific primers (SSP). The effects of HPA compatibility on platelet recovery, incidences of graft-versus-host disease (GVHD) and relapse, and overall survival was investigated. RESULTS: There were no significant differences in platelet recovery according to HPA matching status. We observed no statistically significant differences in the occurrence of relapse and overall survival according to HPA-1, -2, and -3 matched/mismatched groups of patients, whereas HPA-3 mismatching was found to have a significant effect on GVHD development. There was also no difference in GVHD occurrence according to HPA-1 and -2 matched or mismatched transplants. CONCLUSIONS: Since platelet recovery in the HPA-1, -2, -3, and -5 matched/mismatched groups is not significantly different, the seems that platelet glycoprotein (GP) does not seem to act as a factor influencing the homing of hematopoietic stem cells. The finding that HPA-3 incompatibility may be involved in GVHD can be of importance. If a role for HPA-3 as minor histocompatibility antigens is confirmed by additional studies, we can ameliorate the outcome of allogeneic stem cell transplantation by typing of HPA and selecting the most closely related donors.

PCR Based Detection of Phellinus linteus using Specific Primers Generated from Universal Rice Primer (URP) Derived PCR Polymorphic Band

This study was carried out to develop specific primers for PCR detection of Phellinus linteus. Diverse genomes of 15 Phellinus spp. including five Phellinus linteus isolates were fingerprinted by Primer Universal rice primer (URP)1F. The URP-PCR pattern differentiated P. linteus isolates from other phellinus spp. A polymorphic band (2.8 kb), which is unique for P. linteus isolates, was isolated and sequenced. Twenty four-oligonucleotide primer pairs were designed based on information of DNA sequence. The primer set (PLSPF2/PLSPR1) amplified single band (2.2 kb) of expected size with genomic DNA from seven Phellinus linteus, but not with that of other Phellinus species tested. The primers could be used identically in both DNA samples from mycelium and fruit bodies. This specific primers could offer a useful tool for detecting and identifying P. linteus rapidly.

Detection of K-ras mutation in DNA extracted from the plasma of patients with colorectal cancer / 中华消化杂志

Objective The purpose of this study was to determine the value of K-ras mutation in DNA extracted from the plasma as clinical indicator of colorectal cancer. Methods Point mutation at codon 12 of K-ras gene was assayed by polymerase chain reaction of sequence-specific primers in DNA extracted from the plasma and tumors from 32 patients with colorectal cancer. The mutation was further confirmed by dideoxy-mediated chain-termination method of DNA sequencing. Results Fourteen patients (44%) had a codon 12 K-ras mutation within their primary tumors and identical mutations were found in the plasma DNA of 13 patients (93%). Mutant DNA was not detected in the plasma specimens of 18 patients whose tumors tested negative for K-ras alterations or in 5 healthy control subjects. Conclusion Preliminary results suggest the detection of K-ras mutations in circulating DNA extracted from the plasma specimens may have some clinical uti- lity in the detection of colorectal cancer.