369 AMPLIFICATION OF THE SRY GENE FOR SEXING OF BUFFALO (BUBALUS BUBALIS) EMBRYOS USING NESTED MULTIPLEX PCR

2007 ◽  
Vol 19 (1) ◽  
pp. 300
Author(s):  
M. Zhang ◽  
Q. Fu ◽  
W. S. Qin ◽  
H. Y. Zheng ◽  
Y. Q. Lu ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Internal Control ◽  
Pcr Amplification ◽  
Single Copy ◽  
Bubalus Bubalis ◽  
Sex Identification ◽  
Sry Gene ◽  
Embryo Sex ◽  
Pcr Method ◽  
Male Specific

In mammals, the Y chromosome-linked SRY gene is responsible for male sex determination. Therefore, a logical approach for embryo sex identification is to amplify the male-specific single-copy SRY gene. The objective of this study was to design specific primers for amplification of buffalo SRY gene and develop a reliable PCR method for sex identification of buffalo embryos. Genomic DNA was extracted from swamp buffalo (Bubalus bubalis) peripheral blood. A pair of primers based on the sequence of Holstein bovine SRY gene (forward, 52-GTTTGCCTTATGGATTTATT-32; reverse, 52-TCTACTTTAGCCTATTTG-32) was used to amplify whole buffalo SRY gene. This amplified fragment was isolated and constructed into plasmids for sequencing. Two pairs of primers, S1/S2 (forward, 52-CCATGAACGCCTTCATTTTGTG-32; reverse, 52-ACGAGGTCGATATTTATAGC CC-32) and S3/S4 (forward, 52-AAGCAGCTGGGCTATGAGTGGAA-32; reverse, 52-ACGAGGTCGATATTTATAGCCC-32), were designed based on the SRY sequence above. Simultaneously, the G3PDH gene was amplified to serve as an internal control for both male and female embryos. A multiplex-nested-PCR system was optimized by varying the following parameters individually: concentration of Mg2+, dNTPs, primers, and different cycles number. Twenty-seven IVF morulae were identified with the optimal PCR procedure after biopsy. Accuracy of PCR amplification was verified by dot blotting. The sex of 24 embryos fertilized with Y-sperm separated by flow cytometry was also examined. Results indicated that the optimal procedure of Nested-Multiplex-PCR consisted of 1.5 mM Mg2+, 100 �M dNTPs, 0.5 �M SA3/SA4 primers, and 0.25 �M GA3/GA4 primers, and 35 cycles. Accuracy of identification was 100% for 27 IVF morulae; 14 were judged as males and 13 were females. The result of blotting confirmed that the accuracy of amplification was 100%. The proportion of males was 83.3% (20/24) in embryos fertilized with Y-sperm. This confirms that the PCR system targeting the SRY gene can be used for accurate sex identification of buffalo embryos. This study was supported by grants from the Foundation of Guangxi Key Laboratory for Subtropical Bio-Resource Conservation and Utilization (SB0403) and the Guangxi Department of Science and Technology (0626001-3-1).

scholarly journals New Male Specific Markers for Hop and Application in Breeding Program

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andreja Čerenak ◽  
Zala Kolenc ◽  
Petra Sehur ◽  
Simon P. Whittock ◽  
Anthony Koutoulis ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Dna Sequences ◽  
Low Cost ◽  
Pcr Amplification ◽  
Breeding Program ◽  
Phenotypic Data ◽  
World Collection ◽  
Crude Sample ◽  
Male Sex ◽  
Male Specific

Abstract Male specific DNA sequences were selected from a Diversity Arrays Technology (DArT) mapping study to evaluate their suitability for determination of the sex phenotype among young seedlings in a hop (Humulus lupulus L.) breeding program. Ten male specific DArT markers showed complete linkage with male sex phenotype in three crossing families. Following optimization, four were successfully converted into PCR markers and a multiplex PCR approach for their use was developed. Among 197 plants (97 from the world collection; 100 from three segregating families), 94–100% positive correlation with sex phenotypic data was achieved for the single PCR amplification, whereas the multiplex approach showed 100% correlation. To develop a fast and low-cost method, crude sample multiplex PCR was evaluated in 253 progenies from 14 segregating populations without losing accuracy. The study describes, for the first time, the routine application of molecular markers linked to male sex in an intensive Slovenian hop breeding program. The methods described could be employed for screening of sex at the seedling stage in other hop programs worldwide, thereby saving resources for desirable female plants.

scholarly journals A Multiplex-PCR Method for Strain Identification and Detailed Phylogenetic Analysis of AY-Group Phytoplasmas

Plant Disease ◽  
2014 ◽  
Vol 98 (3) ◽  
pp. 299-305 ◽  
Author(s):  
Shigeyuki Kakizawa ◽  
Yoichi Kamagata
Keyword(s):  
Phylogenetic Analysis ◽  
Multiplex Pcr ◽  
16S Rdna ◽  
Pathogenic Bacteria ◽  
Target Genes ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
Single Copy ◽  
Strain Identification ◽  
Specific Detection

Phytoplasmas are plant pathogenic bacteria that cause devastating losses in the yield of diverse crops worldwide. Specific detection and strain identification of phytoplasmas is important to prevent the spread of phytoplasma-induced diseases. Hence, methods to rapidly detect these organisms are important for pest control. Polymerase chain reaction (PCR) methods using phytoplasma-specific primers are widely used to detect phytoplasmas from infected plants and insects because they are highly sensitive, easily handled, and have a variety of analytical secondary applications. The phytoplasma 16S rDNA was widely used as a target of the PCR detection method; however, further target genes and more rapid methods have been required for more specific detection of phytoplasmas. Here, we developed a multiplex-PCR system to amplify several phytoplasma genes. We designed 36 primers, based on the genome sequence of ‘Candidatus Phytoplasma asteris’, to amplify 18 single-copy genes covering wide regions of the phytoplasma genome. Nine genes could be simultaneously amplified in a single PCR. This multiplex-PCR was applied to DNAs from 10 phytoplasma strains belonging to the AY-group, and different amplification patterns were obtained between strains, suggesting that this method would allow us to differentiate phytoplasmas at the strain level. Direct sequencing was also possible after the multiplex-PCR amplification by a modified sequencing method. Detailed phylogenetic analysis was performed using concatenated sequences, and evolutionary relationships among four Japanese isolates were revealed, where these strains could not be distinguished by their 16S rDNA. Thus, this multiplex-PCR system is useful for rapid strain identification and detailed phylogenetic analysis of phytoplasmas.

scholarly journals Development of an Assay for Rapid Detection and Quantification of Verticillium dahliae in Soil

2012 ◽  
Vol 102 (3) ◽  
pp. 331-343 ◽  
Author(s):  
Guillaume J. Bilodeau ◽  
Steven T. Koike ◽  
Pedro Uribe ◽  
Frank N. Martin
Keyword(s):  
Real Time ◽  
Verticillium Dahliae ◽  
Internal Control ◽  
Real Time Pcr ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Single Copy ◽  
Taqman Assay ◽  
Wide Range ◽  
Pathogen Populations

Verticillium dahliae is responsible for Verticillium wilt on a wide range of hosts, including strawberry, on which low soil inoculum densities can cause significant crop loss. Determination of inoculum density is currently done by soil plating but this can take 6 to 8 weeks to complete and delay the grower's ability to make planting decisions. To provide a faster means for estimating pathogen populations in the soil, a multiplexed TaqMan real-time polymerase chain reaction (PCR) assay based on the ribosomal DNA (rDNA) intergenic spacer (IGS) was developed for V. dahliae. The assay was specific for V. dahliae and included an internal control for evaluation of inhibition due to the presence of PCR inhibitors in DNA extracted from soil samples. An excellent correlation was observed in regression analysis (R2 = 0.96) between real-time PCR results and inoculum densities determined by soil plating in a range of field soils with pathogen densities as low as 1 to 2 microsclerotia/g of soil. Variation in copy number of the rDNA was also evaluated among isolates by SYBR Green real-time PCR amplification of the V. dahliae-specific amplicon compared with amplification of several single-copy genes and was estimated to range from ≈24 to 73 copies per haploid genome, which translated into possible differences in results among isolates of ≈1.8 cycle thresholds. Analysis of the variation in results of V. dahliae quantification among extractions of the same soil sample indicated that assaying four replicate DNA extractions for each field sample would provide accurate results. A TaqMan assay also was developed to help identify colonies of V. tricorpus on soil plates.

scholarly journals Molecular Markers for Human Sex Determination in Forensic Genetics Analysis

2021 ◽  
Vol 8 (6) ◽  
pp. 25-30
Author(s):  
Maan H. Salih
Keyword(s):  
Sex Determination ◽  
Multiplex Pcr ◽  
Genetic Marker ◽  
Y Chromosome ◽  
Internal Control ◽  
Forensic Genetics ◽  
Sex Identification ◽  
Pcr Analysis ◽  
Positive Band ◽  
Two Samples

Sex determination is indispensable in forensic anthropology, sexual disorder, and also as part of large-scale genetic population studies. The purpose of this investigation is to determine the human sex from whole blood using multiplex PCR analysis. Blood samples from 75 male and 70 female healthy volunteers were taken from Tikrit city, Iraq. Our study identified a reliable set of three primer locus, namely SRY, ALT1 (internal control) and amelogenin locus. The SRY primer on the Y chromosome showed a 254 bp of PCR product, with 100% accuracy for human male identification. Thus, the pair of SRY primers was considered a strong genetic marker for human sex identification. Amelogenin regions in the Y chromosome showed a true positive band (236 bp) with 100% accuracy on sex identification. Amelogenin regions in X chromosome also showed positive bands (330 bp) in female samples and positive band in male samples except for two samples showed a negative band (null bands). The most obvious finding from this study is that multiplex PCR of ALT1 and SRY is consider as a reliable genetic marker for human sex identification. The research has also shown that amelogenin is good genetic marker for human sex identification.

Establishment of Multiple-PCR for Diagnosing Three Food-Borne Pathogenic Bacteria in Aquatic Products

2012 ◽  
Vol 554-556 ◽  
pp. 1029-1032
Author(s):  
Hui Li Xia ◽  
Hui Zheng ◽  
Jin Jun Zhang
Keyword(s):  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Vibrio Parahaemolyticus ◽  
Pathogenic Bacteria ◽  
Pcr Amplification ◽  
National Standard ◽  
Aquatic Products ◽  
Gene Coding ◽  
Food Borne ◽  
Pcr Method

A rapid multiplex-PCR method was established in order to detect three foodborne pathogenic bacteria including Salmonella typli，Vibrio parahaemolyticus and Listeria monocytogenes in aquatic products. Three pairs of oligonucleotide primers were designed for multiplex-PCR amplification according to gene coding invasion protein A of Salmonella typli，gene coding immune response of Vibrio parahaemolyticus and regulation histone gene of Listeria monocytogenes. The amplified fragment sizes of these three bacteria were 213 bp, 369 bp and 517bp, respectively．The specificity of the multiplex- PCR was high and the minimum detection limit reached 103 CFU/mL. The multiplex-PCR method was used to analyze three aquatic product samples compared with national standard methods, the coincidence rate of two methods reached 100%. The method developed in this study had high sensitivity and specificity, which could be applied for the rapid detection and molecular epidemiology survey of food-borne pathogenic bacteria in aquatic products．

sequences allows for the design of PCR primer sets that specifically amplify the genes encoding the enzyme from DNA samples extracted from the natural environment (Henckel et al. 1999, Meyer et al. 1999, Mesarch et al. 2000). Although the PCR method is both specific and sensitive, such standard reactions are not quantitative. To obtain quantitative data from PCR-based analyses, statistical methods based on most probable number (MPN) estimations have been used (Wand et al. 1997). In MPN-PCR, DNA extracts are diluted before PCR amplification and limits are set on the number of genes in the sample by reference to known control dilutions. Another way to quantify PCR-amplified products for comparison is to include an internal control in the PCR reaction (Leser et al. 1995, Mesarch et al. 2000). Here, a known amount of target DNA is added to a PCR reaction containing DNA from the mixed microbial population. The known target DNA is complementary to the same primers and thus competes with the target sequences in the extracted DNA sample. By preparing a dilution series of the known and unknown DNA species, it is possible to quantify the amount of product produced from die complementary gene in the extracted DNA. The known DNA target can be generated by cloning the gene of interest or purifying the PCR-amplified product after which a deletion is introduced to give a differently sized PCR product. There exists many variations of the standard PCR technique. The sensitivity and specificity of the PCR may be improved by adopting a nested approach. The initial amplification is carried out with a pair of primers that are not organism-specific, whereafter a second round of amplification is conducted on the product using primers specific for an organism with target sites internal to the first primer pair (el Fantroussi et 1997, Levesque et al. 1997, Rheims and Stackebrandt 1999).

2003 ◽  
pp. 223-223
Keyword(s):  
Internal Control ◽  
Pcr Amplification ◽  
Most Probable Number ◽  
Probable Number ◽  
Pcr Product ◽  
Target Dna ◽  
Genes Encoding ◽  
Pcr Method ◽  
Primer Sets ◽  
Nested Approach

A novel common primer multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat species

Food Control ◽  
2009 ◽  
Vol 20 (4) ◽  
pp. 366-370 ◽  
Author(s):  
Weibin Bai ◽  
Wentao Xu ◽  
Kunlun Huang ◽  
Yanfang Yuan ◽  
Sishuo Cao ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Meat Species ◽  
Pcr Method

A PCR assay for sex determination of yak (Bos grunniens) meat by amplification of the male-specific SRY gene

Food Control ◽  
2010 ◽  
Vol 21 (5) ◽  
pp. 726-731 ◽  
Author(s):  
W.L. Bai ◽  
R.H. Yin ◽  
S.J. Zhao ◽  
C. Li ◽  
Z.J. Ma ◽  
...  
Keyword(s):  
Sex Determination ◽  
Sry Gene ◽  
Pcr Assay ◽  
Bos Grunniens ◽  
Male Specific
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