Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens.
01 Feb 1990-Journal of Clinical Microbiology (American Society for Microbiology)-Vol. 28, Iss: 2, pp 276-282
TL;DR: A PCR typing method was devised in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels, which provided a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.
Abstract: The rotavirus gene segment coding for the major outer capsid glycoprotein vp7 was amplified directly from stool specimens by the polymerase chain reaction (PCR). Double-stranded RNA extracted from stool samples was used as the template for reverse transcription, which was followed immediately and in the same reaction mix with amplification, using the Taq polymerase. Various conditions were examined to optimize the yield of the amplified gene. The concentrations of MgCl2, dimethyl sulfoxide, and template RNA were critical. The choice of primer pairs allowed amplification of the entire segment or specific portions. By using type-specific primers derived from distinct regions on the gene, we devised a PCR typing method in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels. The PCR typing method was applied to 10 rotavirus reference strains, including all 6 known human serotypes (serotypes 1, 2, 3, 4, 8, and 9), and to 34 stool specimens previously serotyped by an enzyme immunoassay with monoclonal antibodies. An absolute correlation was found between the molecular and serologic methods. In addition, 14 stool specimens nonserotypable by an enzyme immunoassay with monoclonal antibodies could be typed by the PCR method. Besides the application for rotavirus detection and typing directly from stools, the PCR method provides a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.
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TL;DR: The results suggest that gene 4 typing will be useful in providing more a complete characterization of HRV strains of epidemiologic or vaccine-related interest.
Abstract: Five genetically distinct human rotavirus (HRV) gene 4 groups have been described on the basis of comparative nucleotide sequencing and the predicted amino acid sequences, and at least four of them represent distinct VP4 antigenic types. To identify each gene 4 type and investigate its distribution in HRV isolates from patients with diarrhea, we developed a polymerase chain reaction (PCR) typing method using sequence information available for four genetically distinct gene 4 types. Rotavirus double-stranded RNAs (dsRNAs) isolated from stool samples were first reverse transcribed and amplified by PCR by using two oligonucleotide primers that correspond to regions that are highly conserved among all known HRV gene 4 types. The 876-bp dsDNA products were then reamplified by PCR in the presence of a cocktail containing one conserved plus-sense primer and four type-specific minus-sense primers (selected from the hypervariable region of gene 4), resulting in products of 345, 483, 267, and 391 bp corresponding to gene 4 types 1, 2, 3, and 4, respectively. This method reliably identified the gene 4 types of 16 well-characterized HRV isolates. Our results were independently confirmed for all 16 strains by reverse transcription and PCR amplification of HRV dsRNA in the presence of alternate type-specific primer pairs. For direct gene 4 typing of HRV in stool samples, we developed a method to extract rotavirus dsRNA from stool specimens by using glass powder. Our results suggest that gene 4 typing will be useful in providing more a complete characterization of HRV strains of epidemiologic or vaccine-related interest.
1,343 citations
TL;DR: In infants, natural rotavirus infection confers protection against subsequent infection, which increases with each new infection and reduces the severity of the diarrhea.
Abstract: Background Rotavirus is the leading cause of severe diarrhea in infants. To provide a base line for assessing the efficacy of rotavirus vaccines, we evaluated the protection that is conferred by natural rotavirus infection. Methods We monitored 200 Mexican infants from birth to two years of age by weekly home visits and stool collections. A physician assessed the severity of any episodes of diarrhea and collected additional stool specimens for testing by enzyme immunoassay and typing of strains. Serum collected during the first week of life and every four months thereafter was tested for antirotavirus IgA and IgG. Results A total of 316 rotavirus infections were detected on the basis of the fecal excretion of virus (56 percent) or a serologic response (77 percent), of which 52 percent were first and 48 percent repeated infections. Children with one, two, or three previous infections had progressively lower risks of both subsequent rotavirus infection (adjusted relative risk, 0.62, 0.40, and 0.34, respecti...
860 citations
TL;DR: Strain surveillance helps to determine whether the most prevalent local strains are likely to be covered by the serotype antigens found in current vaccines, and identified globally (G9) or regionally (G5, G8, and P2A[6]) common serotypes not cover by the reassortant vaccines that have undergone efficacy trials.
Abstract: The development of rotavirus vaccines that are based on heterotypic or serotype-specific immunity has prompted many countries to establish programs to assess the disease burden associated with rotavirus infection and the distribution of rotavirus strains. Strain surveillance helps to determine whether the most prevalent local strains are likely to be covered by the serotype antigens found in current vaccines. After introduction of a vaccine, this surveillance could detect which strains might not be covered by the vaccine. Almost 2 decades ago, studies demonstrated that 4 globally common rotavirus serotypes (G1-G4) represent >90% of the rotavirus strains in circulation. Subsequently, these 4 serotypes were used in the development of reassortant vaccines predicated on serotype-specific immunity. More recently, the application of reverse-transcription polymerase chain reaction genotyping, nucleotide sequencing, and antigenic characterization methods has confirmed the importance of the 4 globally common types, but a much greater strain diversity has also been identified (we now recognize strains with at least 42 P-G combinations). These studies also identified globally (G9) or regionally (G5, G8, and P2A[6]) common serotype antigens not covered by the reassortant vaccines that have undergone efficacy trials. The enormous diversity and capacity of human rotaviruses for change suggest that rotavirus vaccines must provide good heterotypic protection to be optimally effective.
598 citations
Cites background from "Polymerase chain reaction amplifica..."
...rotypes by genotyping [31] permitted the detection of both the common serotypes (G1–G4) and the rare serotypes (e....
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TL;DR: The epidemiological data clearly demonstrates that filter feeding bivalve shellfish can, and do, act as efficient vehicles for the transmission of enteric viruses transmitted by the faecal-oral route, and new viral test methods based on PCR, and the development of alternative more reliableFaecal pollution indicators, offer new approaches for the further development of public health controls.
535 citations
TL;DR: Methods and oligonucleotide primers are described to overcome failures to type G9, G 10 and P[11] rotavirus strains, and cross-reactivity identified between G10 and G3 rotaviruses.
411 citations
Cites background or methods from "Polymerase chain reaction amplifica..."
...The sequence alignment of the G10 rotavirus strains mistyped by RT-PCR as G3 showed that the G3-specific primer aET3 (Gouvea et al., 1990) was complementary to the G10 strains in all but two positions (Fig....
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...Reverse transcription with random primers and G and P typing semi-nested PCRs were performed as described previously (Gentsch et al., 1992; Gouvea et al., 1990; Iturriza-Gómara et al., 1999)....
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...2nd round G1 CAA GTA CTC AAA TCA ATG ATG G (nt 314–335) 618 Gouvea et al. (1990 ) G2 CAA TGA TAT TAA CAC ATT TTC TGT G (nt 411–435) 521 Gouvea et al. (1990) G3 ACG AAC TCA ACA CGA GAG G (nt 250–269) 682 This paper G4 CGT TTC TGG TGA GGA GTT G (nt 480–499) 452 Gouvea et al. (1990) G8 GTC ACA CCA TTT GTA AAT TCG (nt 178–198) 754 Gouvea et al. (1990) G9 CTT GAT GTG ACT AYaA AAT AC (nt 757–776) 179 Modified fromGouvea et al....
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...2nd round G1 CAA GTA CTC AAA TCA ATG ATG G (nt 314–335) 618 Gouvea et al. (1990 ) G2 CAA TGA TAT TAA CAC ATT TTC TGT G (nt 411–435) 521 Gouvea et al. (1990) G3 ACG AAC TCA ACA CGA GAG G (nt 250–269) 682 This paper G4 CGT TTC TGG TGA GGA GTT G (nt 480–499) 452 Gouvea et al. (1990) G8 GTC ACA CCA TTT GTA AAT TCG (nt 178–198) 754 Gouvea et al....
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...Reverse transcription with random primers and G and P typing semi-nested PCRs were performed as described previously ( Gentsch et al., 1992; Gouvea et al., 1990; Iturriza-Gómara et al., 1999)....
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References
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TL;DR: A method whereby a nucleic acid sequence can be exponentially amplified in vitro is described in the chapter, and the possibility of utilizing a heat-stable DNA polymerase is explored so as to avoid the need for addition of new enzyme after each cycle of thermal denaturation.
Abstract: Publisher Summary This chapter discusses the specific synthesis of deoxyribonucleic acid (DNA) in vitro through the medium of a polymerase-catalyzed chain reaction. A method whereby a nucleic acid sequence can be exponentially amplified in vitro is described in the chapter. The same method can be used to alter the amplified sequence or to append new sequence information to it. It is necessary that the ends of the sequence be known in sufficient detail that oligonucleotides can be synthesized, which will hybridize to them and that a small amount of the sequence be available to initiate the reaction. The oligonucleotides are complementary to different strands of the desired sequence and at relative positions along the sequence such that the DNA polymerase extension product of the one, when denatured, can serve as a template for the other and vice versa. Oligonucleotides were synthesized using an automated DNA synthesis machine (Biosearch, Inc., San Rafael, California) using phosphoramidite chemistry. “Mispriming"” can be usefully employed to make intentional in vitro mutations or to add sequence information to one or both ends of a given sequence. The chapter explores the possibility of utilizing a heat-stable DNA polymerase so as to avoid the need for addition of new enzyme after each cycle of thermal denaturation
6,055 citations
"Polymerase chain reaction amplifica..." refers background in this paper
...by Mullis and Faloona (22) had been extended for the...
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...Under ideal conditions, PCR can be extremely sensitive, recognizing one copy of a genome (22, 23), but this sensitivity was not achieved in this study....
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TL;DR: D diagnosis of chronic myeloid and acute lymphocytic leukemias by this procedure is rapid, much more sensitive than existing protocols, and independent of the presence or absence of an identifiable Philadelphia chromosome.
Abstract: The Philadelphia chromosome is present in more than 95% of chronic myeloid leukemia patients and 13% of acute lymphocytic leukemia patients. The Philadelphia translocation, t(9;22), fuses the BCR and ABL genes resulting in the expression of leukemia-specific, chimeric BCR-ABL messenger RNAs. To facilitate diagnosis of these leukemias, we have developed a method of amplifying and detecting only the unique mRNA sequences, using an extension of the polymerase chain reaction technique. Diagnosis of chronic myeloid and acute lymphocytic leukemias by this procedure is rapid, much more sensitive than existing protocols, and independent of the presence or absence of an identifiable Philadelphia chromosome.
717 citations
"Polymerase chain reaction amplifica..." refers background in this paper
...detection of single-stranded RNA sequences after copy into cDNA by reverse transcriptase (7, 13, 18)....
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...Thus, the amplified segment does not require further confirmation by hybridization to a specific, labeled probe (18, 23)....
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TL;DR: A surprising observation that emerged from this study was the existence of a rotavirus (porcine strain SB-1A) bridging serotypes 4 and 5, which was identified in both humans and pigs.
Abstract: A total of 16 different strains of rotavirus derived from seven mammalian species (four each from human and porcine species, two each from equine and simian species, and one each from canine and bovine species) and two avian species (one each from turkeys and chickens) were examined in plaque-reduction neutralization tests. Seven antigenically distinct serotypes were established on the basis of a greater than or equal to 20-fold difference between titers of homologous and heterologous reciprocal neutralizing antibodies. Serotypes 1 (strain Wa) and 2 (strain DS-1) were recovered only from humans. Serotype 3 included human rotavirus strain WALK 57/14, rhesus monkey rotavirus strain MMU18006 , vervet monkey rotavirus strain SA-11, dog rotavirus strain CU-1, and horse rotavirus strain H-2. The newly established serotype 4 was identified in both humans (strain St. Thomas no. 4) and pigs (strains Gottfried , SB-1A, and SB-2). Porcine (strain OSU ) and equine (strain H-1) rotaviruses made up a possible fifth serotype. Bovine rotavirus (strain NCDV) constituted a sixth serotype, and chicken rotavirus (strain Ch 2), which had a prime-strain relation with turkey rotavirus (strain Ty 1), was designated serotype 7. A surprising observation that emerged from this study was the existence of a rotavirus (porcine strain SB-1A) bridging serotypes 4 and 5.
422 citations
"Polymerase chain reaction amplifica..." refers background in this paper
...of human rotavirus that have been defined by in vitro neutralization assays (2, 3, 16, 20, 34)....
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TL;DR: Thirty-six monoclonal antibodies immunoprecipitated the 82-kilodalton outer capsid protein, the product of the fourth gene, the viral hemagglutinin, and neutralized rhesus rotavirus to moderate or high titer.
Abstract: A series of monoclonal antibodies was isolated which reacted with one of two major surface proteins of rhesus rotavirus. Thirty-six monoclonal antibodies immunoprecipitated the 82-kilodalton outer capsid protein, the product of the fourth gene, the viral hemagglutinin. These monoclonal antibodies exhibited hemagglutination inhibition activity and neutralized rhesus rotavirus to moderate or high titer. Three monoclonal antibodies immunoprecipitated the 38-kilodalton outer capsid glycoprotein, the eighth or ninth gene product. These three monoclonal antibodies neutralized rhesus rotavirus to high titer and also inhibited viral hemagglutination.
289 citations
"Polymerase chain reaction amplifica..." refers background or methods in this paper
...The PCR technique, originally described for amplification of DNA segments, has been extended for the detection of single-stranded RNA viruses such as the human immunodeficiency virus (12) and rhinovirus (7) following reverse transcription into cDNA copies....
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...Both proteins appear to be involved in virus neutralization (1, 12, 26), but serotype specificity is defined by vp7, which is encoded by gene segment 9 (or segment 8, depending on the strain) (6, 15)....
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