Large-scale use of molecular markers in plant breeding is limited by the throughput capacity for genotyping. DNA polymorphisms can be detected in denaturing polyacrylamide gels indirectly by nucleotide labeling or directly by staining. Fluorescent-labeling or radiolabeling requires sophisticated infrastructure not always available in developing countries. We present an improved low-cost method for silver staining and compare it to 2 other methods for their ability to detect simple sequence repeat polymorphisms in denaturing polyacrylamide gels bound to glass plates. The 3 procedures differed in their requirement for an oxidation pretreatment, preexposure with formaldehyde during silver nitrate impregnation, inclusion of silver thiosulfate, and by their replacement of sodium carbonate for sodium hydroxide to establish alkaline conditions for silver ion reduction. All methods detected the same banding pattern and alleles. However, important differences in sensitivity, contrast, and background were observed. Two methods gave superior sensitivity, detecting down to 1 μL of loaded amplification products. Our improved method gave lower backgrounds and allowed reutilization of staining solutions. The use of thin (<1 mm) denaturing sequencing gels allows genotyping of 60–96 samples within 4 h. Use of smaller loading sample volumes and reutilization of staining solutions further reduced costs.

Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining

A polymerase chain reaction-based assay capable of detecting a broad range of pestiviruses from pigs, cattle, or sheep was developed. Of six sets of primers selected from different parts of the pestivirus genome, the best results were provided by a pair from the highly conserved 5′ non-coding region which gave amplification with all 129 isolates tested. This panel consisted of 33 isolates from pigs, 79 from cattle, and 17 from sheep. Differentiation between the viruses was achieved by cutting the PCR-amplified products with the restriction endonucleases AvaI and Bg1I. Using this procedure it was possible to distinguish at least 3 genogroups; group 1 (HCV) contained 32 of the pig isolates, group II (BVDV) contained all the cattle isolates tested plus 6 sheep isolates and group III (BDV) contained 11 sheep isolates and 1 pig isolate.

Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis

A polymorphic microsatellite (Y-27H39) based on a (GATA)
 n
 repeat was recently discovered on the short arm of the human Y chromosome. We have used a simple technique based on polymerase chain reaction amplification and native polyacrylamide gel electrophoresis followed by highly sensitive silver staining to study the inheritance, the genetic stability and the allele frequency distribution of this polymorphism in the Brazilian population. We have analyzed 100 randomly chosen Caucasian Brazilian father-son pairs with established paternity. Five alleles, four base-pairs apart, were easily distinguishable. Their frequencies were: A (186 bp), 0.19; B (190 bp), 0.49; C (194 bp), 0.24; D (198 bp), 0.07; E (202 bp), 0.01. In all father-son pairs, there was complete allelic concordance. From these data, the probability of discrimination for forensic cases and the average probability of exclusion for paternity cases were both calculated to be 0.66.

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Genetic and population study of a Y-linked tetranucleotide repeat DNA polymorphism with a simple non-isotopic technique.

A method was developed for the purification of rotavirus RNA from fecal extracts in order to permit the sensitive identification of group A rotavirus in fecal specimens by the polymerase chain reaction. Sequential reactions with reverse transcriptase and Taq polymerase with directed primers from rotavirus gene 6 yielded characteristic 259-base-pair fragments that were then visualized by silver stain on a polyacrylamide gel. As few as 500 genomic copies of purified rotavirus RNA could be detected in this manner. However, when the method was applied to fecal samples with added rotavirus virions, inhibition was noted in many of the fecal extracts which were tested. The inhibition could be reversed by dilution of the fecal extract, but sensitivity was also reduced by a corresponding dilutional factor. The inhibition was quantitatively removed by an added step in the extraction process that utilized chromatographic cellulose fiber powder (CF11 powder) to purify the rotavirus RNA during a series of rapid washing and elution steps. After CF11 purification, rotavirus RNA could be detected in experimental fecal samples at dilutions 1,000- to 10,000-fold beyond the detection limits of standard techniques such as enzyme immunoassay and the direct visualization of RNA following polyacrylamide gel electrophoresis. Furthermore, following purification by CF11, rotavirus RNA could be detected in all of seven enzyme-linked immunosorbent assay-positive fecal samples obtained from a child with rotavirus gastroenteritis; when CF11 purification was not performed, rotavirus RNA could be detected in only four of these samples, even after the removal of inhibitors by dilution of the extracts. Large-scale identification of rotavirus in fecal specimens may be possible by use of CF11 purification of viral RNA prior to sequential reactions with reverse transcriptase and Taq polymerase in a modified polymerase chain reaction.

Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions.

Escherichia coli, rotaviruses, and Cryptosporidium parvum are discussed in this review as they relate to enteric disease in calves, lambs, and pigs. These microorganisms are frequently incriminated as causative agents in diarrheas among neonatal food animals, and in some cases different strains or serotypes of the same organism cause diarrhea in humans. E. coli causes diarrhea by mechanisms that include production of heat-labile or heat-stable enterotoxins and synthesis of potent cytotoxins, and some strains cause diarrhea by yet undetermined mechanisms. Rotaviruses and C. parvum induce various degrees of villous atrophy. Rotaviruses infect and replicate within the cytoplasm of enterocytes, whereas C. parvum resides in an intracellular, extracytoplasmic location. E. coli, rotavirus, and C. parvum infections are of concern to producers, veterinarians, and public health officials. These agents are a major cause of economic loss to the producer because of costs associated with therapy, reduced performance, and high morbidity and mortality rates. Moreover, diarrheic animals may harbor, incubate, and act as a source to healthy animals and humans of some of these agents.

Some infectious causes of diarrhea in young farm animals.

A rapid simple technique for the diagnosis of rotavirus has been developed based on the sensitive detection of rotavirus double-stranded RNA genome segments separated in polyacrylamide gels. The method utilizes a recently described ultrasensitive silver stain for polypeptides, which can also detect subnanogram amounts of nucleic acid. The sensitivity of the technique is comparable with that of electron microscopy or enzyme-linked immunosorbent assay.

Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels.

Eight field strains of calf rotavirus from the U.K. were compared by neutralisation tests, using convalescent and hyperimmune antisera. Seven of these strains cross-reacted and were considered to be of one serotype, while the 8th was distinguished by a greater than 20-fold two-way difference in neutralisation titre suggesting a second serotype. Three widely-distributed reference strains (U.K., Northern Ireland and Lincoln) cross-reacted with the strains in the dominant serotype, as did 33 of 42 other field calf rotavirus strains. Nine field strains failed to cross-react with either serotype, suggesting the existence of other potential serotypes in the calf population.

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A. J. Herring

Papers

Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels.

Evidence for serotypic variation among bovine rotaviruses.